Engineered dnase enzymes and use in therapy

ABSTRACT

The present invention provides engineered DNase proteins (including DNase1-like 3 and DNase1) that are useful for treating conditions characterized by neutrophil extracellular trap (NET) accumulation and/or release. In some aspects, the invention provides compositions and methods for preventing or treating vascular occlusion involving NETs. As demonstrated herein, NETs participate in a non-canonical mechanism for vascular occlusion, which is not dependent on fibrin or platelets.

PRIORITY

This Application claims the benefit of, and priority to, U.S.Application No. 62/611,166 filed Dec. 28, 2017 and 62/547,220 filed Aug.18, 2017, each of which is hereby incorporated by reference in itsentirety.

BACKGROUND OF THE INVENTION

Inflammation is an essential host response to control invading microbesand heal damaged tissues. Uncontrolled and persistent inflammationcauses tissue injury in a plethora of inflammatory disorders.Neutrophils are the predominant leukocytes in acute inflammation. Duringinfections neutrophils generate neutrophil extracellular traps (NETs),lattices of DNA-filaments decorated with toxic histones and enzymes thatimmobilize and neutralize bacteria. The relevance of NETs in hostdefense is illustrated by the fact that extracellular DNases serve asvirulence factors in several pathogenic bacteria. However,inappropriately released NETs may harm host cells due to theircytotoxic, proinflammatory, and prothrombotic activity. Indeed, NETs arefrequently associated with inflammatory or ischemic organ damage and thetherapeutic infusion of DNases limits host injury in various animalmodels.

Extracellular trap (ET) formation is not limited to neutrophils as otherleukocytes, namely monocytes, macrophages, basophils, eosinophils, andmast cells release ETs as well. Furthermore, cancer cells, includingacute promyelocytic leukemia (APL) cells, and injured endothelial cellscan expose DNA-filaments, which have ET-like features.

How the host degrades NETs in vivo to limit tissue damage duringepisodes of inflammation is poorly understood. DNase1 (D1) in serum hasbeen shown to digest the DNA-backbone of NETs in vitro. Otherextracellular DNases have been identified, including DNase1-like 3(D1L3) in circulation, which can digest extracellularmicroparticle-associated chromatin.

D1 and D1L3 form along with DNase1-like 1 (D1L1) and DNase1-like 2(D1L2) the DNase1-protein family, a group of homologous secreted DNaseenzymes that are expressed in humans and provide drug candidates forNET-associated diseases.

However, the physical, enzymatic, and pharmacokinetic properties ofthese enzymes are not ideal for therapy.

The present invention provides engineered DNases, including DNase1-like3 and DNase1, for therapy, including for preventing or treating vascularocclusions that can result from release of NETs during acuteinflammatory episodes.

SUMMARY OF THE INVENTION

The present invention provides engineered DNase proteins (includingDNase1-like 3 and DNase1) that are useful for treating conditionscharacterized by neutrophil extracellular trap (NET) accumulation and/orrelease. In some aspects, the invention provides compositions andmethods for preventing or treating vascular occlusion involving NETs. Asdemonstrated herein, NETs participate in a non-canonical mechanism forvascular occlusion, which is not dependent on fibrin and platelets.

In some aspects, the invention provides DNase1-like 3 (D1L3) variantsengineered to have physical, pharmacodynamic, and/or enzymaticproperties more suitable for therapy, for example, to reduce or preventNET accumulation in a subject. In some embodiments, the D1L3 enzyme hasadvantages in manufacturing, providing for production of the recombinantenzyme suitable for use in therapy. In various embodiments, theinvention provides a recombinant D1L3 variant comprising one or moreamino acid alterations resulting in one or more glycosylations,inactivation of a nuclear localization signal, and/or deletion of all orpart of a C-terminal tail.

In various embodiments, the D1L3 protein variant has increased proteinstability, increased resistance towards inhibition by proteases,increased bioavailability, and substantially the same or better DNAand/or chromatin and/or NET-degrading activity (in vitro or in vivo) ascompared to wild-type D1L3 protein. In various embodiments, the D1L3variant comprises one or more of the following properties relative towild type D1L3 (e.g., SEQ ID NO:2): the same or substantially the same(or higher) protein-free DNA (naked DNA) degradation activity, the sameor substantially the same (or higher) chromosomal DNA (chromatin)degradation activity, protease resistance, an increased circulatoryhalf-life, and higher production levels with in vitro expression systems(e.g. Chinese hamster ovary cells and/or Pichia pastoris).

In some embodiments, the D1L3 variant includes one or a plurality ofblock substitutions from human DNase1 (SEQ ID NO:1), described herein asbuilding block substitutions.

In some aspects, the invention provides DNase1 (D1) variants engineeredto have physical, pharmacodynamic, and/or enzymatic properties moresuitable for therapy, for example, to reduce or prevent NET accumulationin a subject. In some embodiments, the D1 enzyme has advantages inmanufacturing, providing for production of the recombinant enzymesuitable for use in therapy. In various embodiments, the engineered D1variant comprises an amino acid sequence that is at least 80% identicalto the mature enzyme of amino acid sequence of SEQ ID NO:1, with one ormore amino acid substitutions, additions (including insertions), ordeletions resulting in one or more of a mutated DNA binding site, amutated chromatin binding site, a mutated actin binding site, a mutatedglycosylation site, addition of a nuclear localization signal (e.g.,having similarity or identity to NLS1 or NLS2 of D1L3), and/or aC-terminal domain similar to or identical to the C-terminal tail ofD1L3.

In various embodiments, the D1 variant is engineered to comprise one ormore of the following characteristics relative to the wild type enzyme:the same or higher protein-free DNA (naked DNA) degradation activity,higher chromosomal DNA (chromatin) degradation activity, similar orimproved protease resistance, actin resistance, similar or improvedpenetration from blood into urine and/or bile, and higher productionlevels in vitro expression systems (e.g. Chinese hamster ovary cellsand/or Pichia pastoris).

D1 variants described herein may have a combination of point mutations,including addition of cationic residues, and/or may comprise one or moreblock substitutions from D1L3 (SEQ ID NO:2). Such block substitutionsare described herein, and termed “building block substitutions.”

In some aspects, the invention provides pharmaceutical compositionscomprising the D1L3 and/or D1 variants, or polynucleotides or vectorsencoding the same, and a pharmaceutically acceptable carrier. In variousembodiments, the composition is formulated for parenteral or pulmonaryadministration. In some embodiments, the composition is formulated forintravenous, intraarterial, intraperitoneal, intraarticular,intramuscular, topical, or subcutaneous administration. In someembodiments, the composition comprises both D1L3 and D1, which are eachoptionally engineered variants described herein. In some embodiments,the composition comprises a chimeric protein containing block sequencesfrom both D1 and D1L3. Exemplary block substitutions between D1 and D1L3are shown in FIG. 31.

In still other aspects, the invention provides a process for DNaseenzyme engineering, by producing chimeric sequences. For example, inthese aspects the method comprises providing a protein-protein alignmentof donor and recipient DNase enzymes; identifying variable amino acidsequences for transfer (“building block”). The variable amino acid oramino acids are flanked by one or more conserved amino acids in thedonor and recipient DNase enzymes (upstream and downstream of thebuilding block). These building blocks can be swapped between receipientand donor proteins. The chimeric enzyme is then produced recombinantly.

In still other aspects, the invention provides a method of making apharmaceutical composition for reducing or preventing neutrophilextracellular trap (NET) accumulation. In these embodiments, theinvention employs a genetically modified mouse deficient in D1 and D1L3activity, and heterologous expression of G-CSF polynucleotide (e.g., inhepatocyte cells) or inducing a sustained endogenous G-CSF expression(e.g. via repetitive administration of microbial compounds). This mousemodel accumulates NETs and rapidly develops NET-related vascularocclusions. In these embodiments, the invention comprises administeringa candidate NET inhibitor or candidate DNase enzyme (including a D1L3 orD1 variant in accordance with this disclosure) to thegenetically-modified mouse, and selecting a NET inhibitor or DNaseenzyme that reduces the accumulation of NETs. The selected inhibitor orenzyme is formulated for administration to a human patient.

In some aspects, the invention provides a method for treating a subjectin need of neutrophil extracellular trap (NET) degradation. The methodcomprises administering a therapeutically effective amount of a D1L3and/or D1, and/or variants thereof according to this disclosure. TheD1L3 or D1 variant may be administered as pharmaceutical compositionscomprising the recombinant protein, or in some embodiments the encodingDNA or RNA, or in some embodiments, vectors comprising the same.

In various embodiments, the present invention pertains to the treatmentof diseases or conditions characterized by the presence or accumulationof NETs. Such diseases or conditions include, but are not limited to,diseases associated with chronic neutrophilia (e.g., an increase in thenumber of neutrophils), neutrophil aggregation and leukostasis,thrombosis and vascular occlusion (e.g. sickle cell disease),ischemia-reperfusion injury (e.g. midgut volvulus, testicular torsion,limb ischemia reperfusion, vital organ ischemia-reperfusion, organtransplantation), surgical and traumatic tissue injury, an acute orchronic inflammatory reaction or disease, an autoimmune disease (e.g.systemic lupus erythematosus (SLE), lupus nephritis, rheumatoidarthritis, vasculitis, systemic sclerosis), cardiovascular disease(e.g., myocardial infarction, stroke, atherosclerosis, venousthromboembolism, including thrombolytic therapy), metabolic disease(e.g., diabetes), systemic inflammation (e.g., systemic inflammatoryresponse syndrome (SIRS), sepsis, septic shock, disseminatedintravascular coagulation (DIC), and thrombotic microangiopathy (TMA)),inflammatory diseases of the respiratory tract (e.g. cystic fibrosis,chronic obstructive pulmonary disease (COPD), acute lung injury (ALI),smoke induced lung injury, transfusion induced lung injury (TRALI),acute respiratory distress syndrome (ARDS), and asthma, atelectasis,bronchitis, empyema), renal inflammatory diseases (acute and chronickidney diseases, including acute kidney injury (AM) and chronic kidneydisease (CKD), inflammatory diseases related to transplated tissue (e.g.graft-versus-host disease) and cancer (e.g. leukemia, tumor metastasis,and solid tumors).

In some embodiments, the subject has or is at risk of a ductal occlusionin a ductal system. Non-limiting examples of a ductal system or an organor tissue containing a ductal system include bile duct, tear duct,lactiferous duct, cystic duct, hepatic duct, ejaculatory duct, parotidduct, submandicular duct, major sublingual duct, bartholin's duct,cerebral aqueduct, pancreas, mammary gland, vas deferens, ureter,urinary bladder, gallbladder, and liver. As such, the present inventionis useful for treating a subject who has pancreatitis, cholangitis,conjunctivitis, mastitis, dry eye disease, an obstruction of the vasdeferens, or renal disease.

In other embodiments, the subject has or is at risk of NETs accumulatingon endothelial surfaces (e.g., surgical adhesions), the skin (e.g.,wounds/scarring, uclers), or in synovial joints (e.g., gout, arthritis).For instance, NETs may contribute to surgical adhesions, e.g., after aninvasive medical procedure. The present invention can be administeredduring surgery to prevent or inhibit the formation of surgicaladhesions. In some instances, D1, D1L3 (including variants thereof), orthe combination of D1 and D1L3 (or variants thereof) as outlined hereincan be administered topically to the skin to prevent or treat woundsand/or scarring. In other instances, D1, D1L3, or variants, or thecombinations thereof as outlined herein can be administered to synovialjoints to prevent or treat gout and arthritis.

In some embodiments, the subject has or is at risk of a vascularocclusion comprising NETs.

In various embodiments, the present invention pertains to the treatmentof diseases that are treatable with DNase enzyme, including D1 andstreptodornase. Such diseases or conditions include thrombosis, stroke,sepsis, lung injury, atherosclerosis, viral infection, sickle celldisease, myocardial infarction, ear infection, wound healing, liverinjury, endocarditis, liver infection, pancreatitis, primary graftdysfunction, limb ischemia reperfusion, kidney injury, blood clotting,alum-induced inflammation, hepatorenal injury, pleural exudations,hemotorax, intrabiliary blood clots, post pneumatic anemia, ulcers,otolaryngological conditions, oral infections, minor injuries,sinusitis, post-operative rhinoplasties, infertility, bladder catheter,wound cleaning, skin reaction test, pneumococcal meningitis, gout, legulcers, cystic fibrosis, Kartegener's syndrome, asthma, lobaratelectasis, chronic bronchitis, bronchiectasis, lupus, primary cilliarydyskinesia, bronchiolitis, empyema, pleural infections, cancer, dry eyesdisease, lower respiratory tract infections, chronic hematomas,Alzheimer's disease, and obstructive pulmonary disease.

In still other embodiments, the invention provides a method forrecombinant production of D1 or D1L3, or variants thereof, using anon-mammlian expression system, such as Pichia pastorisIn someembodiments, the Pichia pastoris encodes the DNase enzyme with thenative signal peptide allowing for secretion from host cells.

Other objects, embodiments and advantages of the present invention areapparent in the Detailed Description below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-FIG. 1F illustrate that DNase1 and DNase1l3 in circulationdegrade NETs in vitro, and characterize DNA-degrading activity of serafrom WT, DNase1−/− (D1−/−), DNase1l3−/−(D1L3−/−),DNase1/DNase1l3−/−(D1/D1L3−/−) mice. FIG. 1A provides detection ofDNase1 (D1), DNase1l3 (D1L3), and total DNase activity by thezymographic assays DPZ and SRED. FIG. 1B provide images and FIG. 1Cprovide quantification of DNA-stainings of NETs generated in vitro afterincubation with sera from indicated genotypes (N=6). Scale bar: 50 μm.FIG. 1C shows DPZ and SRED analysis of sera from D1/D1L3−/− mice stablyexpressing a plasmid with DNase1 (D1), DNase1l3 (D1L3), or a controlplasmid (Ctrl). FIG. 1E shows images and FIG. 1F shows quantification ofDNA-staining of NETs generated in vitro after incubation with buffer orsera from D1/D1L3−/− mice expressing D1, D1L3, or Ctrl (N=5). Scale bar:50 μm. Images are representative of two or more independent experiments.Statistics: (FIG. 1C and FIG. 1F) one-way ANOVA followed by Bonferroni'smultiple comparisons post test; § P<0.001 vs. all other groups.

FIG. 2A-FIG. 2I illustrate that DNase1 or DNase1l3 is required totolerate chronic neutrophilia. Chronic neutrophilia was induced byinjection of a G-CSF-expression plasmid (CSF3). Controls received anempty plasmid (Ctrl). FIG. 2A shows blood neutrophil count of WT miceand controls after 2 weeks (N=3-6). FIG. 2B shows NET-like structures(arrow) in DNA-stainings of blood smears from WT mice after 1 week(N=3). Scale bar: 20 μm. FIG. 2C shows survival of WT (N=7), DNase1−/−(D1−/−, N=6), DNase1l3−/− (D1L3−/−, N=6), DNase1/DNase1l3−/− mice(D1/D1L3−/−, N=6) and controls (Ctrl, N=4). FIG. 2D shows survival ofD1/D1L3−/− mice co-expressing CSF3 with DNase1 (CSF3/D1, N=5), DNase1l3(CSF3/D1L3, N=6), and controls (CSF3/Ctrl, N=4). FIGS. 2E-2Icharacterizes mortality during chronic neutrophilia (N=4). FIG. 2E showschange in peripheral body temperature. FIG. 2F provides photographs ofplasma and urine. FIG. 2G provides concentration of hemoglobin in blood.FIG. 2H provides images of blood smears. Arrows point to schistocytes.Scale bar: 20 μm. FIG. 21 shows LDH concentration in plasma. UT:untreated mice (N=5-6). Images are representative of three or more mice.Statistics: (FIGS. 2A, 2H, 2I) one-way and (FIG. 2E) two-way ANOVAfollowed by Bonferroni's multiple comparisons post test, (FIG. 2B)student's t-test, (FIGS. 2C, 2D) Log-rank test; #P<0.01, § P<0.001 vs.all other groups or baseline (BL).

FIG. 3A-FIG. 3G illustrate that DNase1 and DNase1l3 prevent vascularocclusion by NET-clots during chronic neutrophilia. Histologicalanalysis of DNase1/DNase1l3−/− mice (D1/D1L3−/−) co-expressing CSF3 withDNase1 (CSF3/D1, N=4), DNase1l3 (CSF3/D1L3, N=4), or a control plasmid(CSF3/Ctrl, N=4). FIG. 3A provides hematoxylin and eosin stainings (H&E)of lungs. Scale bars: 500 μm (overview), 25 μm (detail). FIG. 3B showsquantification of blood vessels in lungs occluded byhematoxylin-positive clots per field of view (FOV). Untreated WT mice(UTWT, N=4), untreated D1/D1L3−/− mice (UT; N=4). FIG. 3C showsimmunostaining of occluded blood vessels for the neutrophil-markermyeloperoxidase (MPO) and chromatin. FIGS. 3D and 3E show immunostainingfor von Willebrand factor (vWF), fibrin, and DNA. Three types ofNET-clots were detected (α, vWF+/fibrin−; β, vWF+/fibrin+; γ,vWF−/fibrin−). FIG. 3F provides quantification of vWF and fibrin inNET-clots. Data shown as mean±SD, n=108. FIG. 3G shows survival ofD1/D1L3−/− mice expressing CSF3 treated with IgG (N=4),anti-platelet-IgG (N=5), and dabigatran (N=5). Scale bars: 50 μm. Imagesare representative of three or more mice. (FIGS. 3C, 3D, 3E) dotted lineindicates vessel wall. Statistics: (FIG. 3B) one-way ANOVA followed byBonferroni's multiple comparisons post test; § P<0.001 vs. all othergroups, (FIG. 3G) Log-rank test; P>0.05 Anti-Platelet-IgG or Dabigatranvs. IgG.

FIG. 4A-FIG. 4I illustrate that DNase1 and DNase1l3 protect against hostinjury in septicemia. WT mice (N=5) and mice with a combined deficiencyin DNase1 and DNase1l3 (D1/D1L3−/−) expressing DNase1 (D1, N=7),DNase1l3 (D1L3, N=8), or a control plasmid (Ctrl, N=11) were treatedwith lipopolysaccharide and heat-killed E. coli to induce septicemia.FIG. 4A shows survival time of septic mice. FIG. 4B shows concentrationof hemoglobin in blood. FIG. 4C shows photographs of plasma and urine.FIG. 4D provides LDH concentration in plasma. FIG. 4E showsquantification of schistocytes in blood per field of view (FOV). FIG. 4Fshows quantification of occluded blood vessels in lungs per FOV. FIG. 4Gshows hematoxylin and eosin stainings (H&E) of lungs. Arrowheads pointto occluded blood vessels. Scale bars: 500 μm. FIGS. 4H and 4I show H&Estaining of partially and fully occluded blood vessel. Arrows point toNETs covering the intercellular space. Inserts are overviews. Scalebars: 50 μm. Statistics: (FIG. 4A) Log-rank test; #P<0.01 vs. all othergroups, (FIGS. 4B-4F) one-way ANOVA followed by Bonferroni's multiplecomparisons post test; § P<0.001, #P<0.01.

FIG. 5A-FIG. 5E show a murine model of chronic neutrophilia. Four-weekold WT mice were injected with a CSF3-expression plasmid (CSF3) or withan empty control plasmid (Ctrl). FIG. 5A shows G-CSF levels in plasma(N=3-6). BL: baseline, before injection; Ctrl: mice expressing thecontrol plasmid without CSF3 for 2 weeks (N=4-5). FIG. 5B showsimmunostaining for neutrophil elastase of lungs, kidneys, and livers.Scale bars: 200 μm. FIG. 5C shows photographs and weights of spleens(N=5-6). Scale bar: 1 cm. FIG. 5D shows body weight of mice at indicatedtimes (N=3-5). FIG. 5E shows neutrophil counts in whole blood and plasmalevels of ALT, AST, BUN, and creatinine 2 weeks after the injection.Images are representative of three or more mice. Statistics: (FIGS. 5A,5C) one-way and (FIG. 5D) two-way ANOVA followed by Bonferroni'smultiple comparisons post test, and (FIG. 5E) Student's t-test; #P<0.01,§ P<0.001 vs. (FIG. 5A) BL, or (FIG. 5C) UT.

FIG. 6A-FIG. 6D show that DNase1 and DNase1l3 prevent the formation ofNET-clots and organ injury in liver and kidneys during chronicneutrophilia. Analysis of DNase1/DNase1l3−/− mice (D1/D1L3−/−)co-expressing CSF3 a control plasmid (CSF3/Ctrl, N=4), with DNase1(CSF3/D1, N=4), or with DNase1l3 (CSF3/D1L3, N=4). Controls includeuntreated WT mice (UTWT, N=4) and untreated D1/D1L3−/− mice (UT; N=4).FIG. 6A shows plasma levels of alanine and aspartate aminotransferases(ALT, AST). FIG. 6B shows plasma levels of blood urea nitrogen (BUN) andcreatinine. FIG. 6C provides quantification of intravascularhematoxylin-rich clots in livers per section and in kidneys per field ofview (FOV). FIG. 6D provides H&E stainings of livers and kidneys. Scalebars: 500 μm (overviews), 50 μm (detail). Images are representative ofthree or more mice. Statistics: (FIGS. 6A, 6B, 6C) one-way ANOVAfollowed by Bonferroni's multiple comparisons post test; #P<0.01, §P<0.001 vs. all other groups.

FIG. 7A-FIG. 7C show hematoxylin-positive clots are composed of NETs.Analysis of lungs from DNase1/DNase1l3−/− mice (D1/D1L3−/−) with chronicneutrophilia (D1/D1L3−/−+CSF3/Ctrl) shown in FIGS. 3A-3G. FIG. 7Aprovides staining with hematoxylin and eosin (H&E), for DNA, and forchromatin of consecutive lung sections. Scale bars: 200 μm. FIG. 7Bprovides immunostaining of occluded blood vessel for chromatin, CRAMP(cathelin-related antimicrobial peptide), and DNA. Scale bars: 50 μm.FIG. 7C provides immunostaining of occluded blood vessel for chromatin,citrullinated histone 3 (citH3), and DNA. Scale bars: 50 μm. Images arerepresentative of three or more mice.

FIG. 8A and FIG. 8B show that human NETs form clots in vitro. FIG. 8Aprovides a photograph of NET-clot (arrow) generated by PMA-activatedhuman neutrophils in vitro. Untreated neutrophils (UT) or neutrophilsactivated in the presence of recombinant human DNase1 (PMA+D1) served ascontrols. Scale bars: 5 mm. FIG. 8B provides hematoxylin and eosinstainings (H&E) and immunostaining for chromatin and theneutrophil-marker myeloperoxidase (MPO) of in vitro generated NET-clots.Scale bars: 200 μm. Images are representative of three or moreindependent experiments.

FIG. 9A and FIG. 9B show NET-degradation correlates with diseaseactivity in STEC-HUS. FIG. 9A shows images and FIG. 9B showsquantification of DNA-staining of NETs generated in vitro afterincubation with buffer, plasma from normal healthy donors (N=7), orplasma collected in the acute disease state (Acute) and in remission(Rem.) of 11 patients with STEC-HUS. Scale bar: 50 μm. Statistics:one-way ANOVA followed by Bonferroni's multiple comparisons post test; §P<0.001 vs. all other groups.

FIG. 10 provides a table of descriptions of human tissue from variouspatients examined, such as patient number, cause of death as indicatedin the autopsy report (ARDS, acute respiratory distress syndrome). Sexand age of the patient. Type of analyzed tissue. The tissues wasanalyzed in a three step procedure: first, identification ofhematoxylin-rich clots/aggregates in hematoxylin & eosin stainings;second, identification of NETs in hematoxylin-rich clots byimmunostaining for chromatin & myeloperoxidase (MPO); third,identification of the localization of the NET-clots within the lungarchitecture.

FIG. 11A-FIG. 11D show human NETs form clots in situ. Lung tissuecollected at autopsy from two patients with sepsis (Patient #6 and #7 inFIG. 10). FIG. 11A shows H&E staining of two hematoxylin-richintravascular clots in lung tissue from patient #6. FIG. 11B showsimmunostaining of consecutive section for chromatin and myeloperoxidase(MPO) of left blood vessel shown in panel A. FIG. 11C shows H&E stainingblood vessel in lung tissue from patient #7 shows hematoxylin-richintravascular clots. FIG. 11D shows immunostaining of consecutivesection for chromatin and MPO. Arrows point to clots of NETs. Scale bar:50 μm.

FIG. 12 shows NET-clots occlude blood vessels in septic mice.Immunostaining of fully occluded blood vessel in septicDNase1/DNase1l3−/− mice for DNA, chromatin and myeloperoxidase (MPO).DNA and chromatin stainings of NETs (arrows) appear dimmer than nuclearstainings (asterisks) and cover the intercellular space. Scale bars: 10μm.

FIG. 13A-FIG. 13F shows that DNase1 and DNase1l3 degrade NETs bydistinct mechanisms. FIG. 13A provides DNA-stainings of PFA-fixed NETsgenerated in vitro after incubation with sera from WT, DNase1−/−(D1−/−), DNase1l3−/− (D1L3−/−), DNase1/DNase1l3−/− mice (D1/D1L3−/−).Data are representative of ≥3 independent experiments. Scale bar: 50 μm.FIG. 13B shows the concentration of cell-free DNA fragments releasedfrom PFA-fixed NETs after incubation with sera from WT, D1−/−, D1L3−/−,and D1/D1L3−/− mice. FIG. 13C shows DNA-stainings of non-fixed NETsgenerated in vitro after incubation with vehicle, recombinant murineDNase1 (rmD1), or recombinant murine DNase1l3 (rmD1L3). FIG. 13D showsthe concentration of cell-free DNA fragments released from non-fixedNETs generated in vitro after incubation with vehicle, rmD1, or rmD1L3.FIG. 13E shows DNA-stainings of PFA-fixed NETs generated in vitro afterincubation with vehicle, rmD1, or rmD1L3. FIG. 13F shows theconcentration of cell-free DNA fragments released from PFA-fixed NETsgenerated in vitro after incubation with vehicle, rmD1, or rmD1L3.Statistics by one-way ANOVA followed by Bonferroni's multiplecomparisons post test; § P<0.0001.

FIG. 14A-FIG. 14G provide DNase1 and DNase1l3 activity in thecirculation of humans and mice. FIG. 14A-FIG. 14E show DNase activity inhuman and murine plasma quantified by SRED. FIG. 14A illustratessupplementation of plasma from normal healthy donors (NHD) withpolyclonal antibodies against human DNase1 (α-hDNase1), but not withIgG, blocks the DNA degrading activity in a concentration-dependentmanner (*: p<0.05 vs. IgG). FIG. 14B shows plasma supplemented with 200μg/ml α-hDNase1 and heparin, an inhibitor of DNase1l3, blocks residualDNase activity in plasma supplemented with 200 μg/ml α-hDNase1 (*:p<0.05 vs. NHD). FIG. 14C-FIG. 14E provide a comparison of DNaseactivity in serial dilutions of plasma from mice and NHD. FIG. 14Cillustrates that total DNase activity of WT mice is approximately10-times higher than in NHD. FIG. 14D shows DNase1 activity in plasmafrom DNase1l3−/− mice is approximately 10-fold higher than human plasmasupplemented with 500 μ/ml heparin. (E DNase1l3 activity in plasma fromDNase1−/− mice is approximately 10-fold higher than human plasmasupplemented with 200 μg/ml α-hDNase1. (F, G) In vitro NET-degradationis illustrated in FIG. 14F and FIG. 14G. FIG. 14F shows activatedneutrophils incubated with plasma from NHD alone or supplemented with200 μg/ml α-hDNase1, plasma from TMA patients, and plasma from WT,DNase1−/−, and DKO mice (Scale bars: 200 μm). FIG. 14G shows cell-freeDNA in supernatants of activated neutrophils incubated with plasma fromindicated sources (§: p<0.001 vs. NHD). In vitro NET-degradation byhuman plasma is dependent on DNase1 as DNase1l3 in human plasma is notsufficient to degrade NETs.

FIG. 15A-FIG. 15C show a lethal course of chronic neutrophilia in analternative strain of DNase1/DNasel3−/− mice. An alternative strain ofDNase1−/− mice (see Methods of Example 1) was crossed with DNase1l3−/−mice to generate mice with a combined deficiency. FIG. 15A providessurvival curves of neutrophilia in WT (N=9), DNase1−/− (D1−/−, N=5), andDNase1/DNase1l3−/− mice (D1/D1L3−/−, N=5). Chronic neutrophilia wasinduced by injection of a CSF3-expression plasmid (CSF3). FIG. 15Bprovides hematoxylin and eosin stainings (H&E) of lung sections. Arrowpoints to hematoxylin positive clots in D1l/D1L3−/− mice. Scale bar: 500μm. FIG. 15C provides survival of D1/D1L3−/− mice coexpressing CSF3 witha control plasmid (CSF3/Ctrl, N=6), with DNase1 (CSF3/D1, N=5), or withDNase1l3 (CSF3/D1L3, N=6). Statistics: Log-rank test; § P<0.001.

FIG. 16A-FIG. 16B show the screening approach to identify hyperactivevariants of DNase1 and DNase1L3 in vitro. FIG. 16A shows a library ofDNases included wild-type DNase1L3 (I, D1L3), wild-type DNase1 (II, D1),and variants thereof. D1 and D1L3 variants were designed by transferringamino acids from D1L3 to D1 (III, D1^(v)) and D1 to D1L3 (IV, D1L3^(v)).DNase activity was tested using the degradation of high-molecular weight(HMW) DNA, dsDNA and chromatin, as a substrate. In FIG. 16B, zymographyshowed dsDNA degrading activity as dark circles. The dsDNA degradingactivity correlates with the diameter. Samples without activity show theloading well as small black spot (e.g. D1, 0.0005 ng). D1 degrades dsDNAapproximately 100-fold more efficiently than D1L3. Agarose gelelectrophoresis (AGE) of DNA isolated from digested chromatin shows ashift from high-molecular weight DNA to lower or low-molecular weightDNA that correlates with chromatin degrading activity. D1L3 degradeschromatin approximately 100-fold more efficiently than D1. At a ratio of10:1 or 1:1 (m/m), D1 and D1L3 show synergistic effects in thedegradation of chromatin.

FIG. 17 shows an amino acid sequence alignment of human DNase1 (SEQ IDNO: 1) and human DNase1L3 (SEQ ID NO: 2). The signal peptide, conservedamino acids, additional arginine- and lysine-residues in variableregions of D1L3, and the C-terminal tail of D1L3 are highlighted.Sequence rulers indicate the amino acid position.

FIG. 18 shows a list of arginine- and lysine-residues that are presentin DNase1L3 only. The corresponding amino acid in DNase1 and amino acidsubstitution in DNase1 are indicated. Numbers in brackets are the aminoacid position in the mature protein without signal peptide. Arginine-and lysine-residues that are located in the C-terminal tail of D1L3 areshown in lanes 29-37. This application tested 22 of the 37 possibleDNase1 variants.

FIG. 19 shows the characterization of DNase1 variants with singlearginine and lysine amino acid substitutions listed in FIG. 18.Zymography showed dsDNA degrading activity as dark circles. The dsDNAdegrading activity correlates with the diameter. Agarose gelelectrophoresis (AGE) of DNA isolated from digested chromatin shows ashift from high-molecular weight DNA to lower or low-molecular weightDNA that correlates with chromatin degrading activity. Amino acidsubstitutions that cause an increase in chromatin degrading activity arehighlighted in blue. Samples without such effect are shown in lightblue.

FIG. 20 shows the amino acid sequence alignment of human DNase1 (SEQ IDNO: 1), mouse DNase1 (SEQ ID NO: 3), rat DNase1 (SEQ ID NO: 4), andhuman DNase1L3 (SEQ ID NO: 2). Sequence rulers indicate the amino acidposition. The signal peptides are highlighted in grey. Cationic aminoacid residues (arginine: R, lysine: K) that are present in mouse/ratDNase1 and human DNase1L3, but not in human DNase1, are highlighted.Four such R/K-residues are shown in mouse DNase1 (Q31R, Q49K, Q79R,W209R). Rat DNase1 contains two additional R/K-residues (Q177K, G262K).

FIG. 21A-FIG. 21B shows a comparison between murine and human DNase1.(FIG. 21A) Zymography show dsDNA degrading activity as dark circles. ThedsDNA degrading activity correlates with the diameter. Samples withoutactivity show the loading well as small black spot (e.g. Ctrl). MurineDNase1 (mD1) and human DNase1 (D1) show similar levels of dsDNAdegrading activity. Agarose gel electrophoresis (AGE) of DNA isolatedfrom digested chromatin shows a shift from high-molecular weight DNA tolower or low-molecular weight DNA that correlates with chromatindegrading activity. Murine DNase1 (mD1) shows more chromatin degradingactivity than human DNase1 (D1). (FIG. 21B) Gene therapy with humanDNase1 provides only partial protection from lethal vascular occlusionby NETs. Chronic neutrophilia with concomitant intravascularNET-formation was induced as by hepatic expression of Csf3, whichencodes with G-CSF. The data show the survival curve of Csf3-expressingDnase1^(−/−)Dnase1l3^(−/−) mice. Animals were injected with Csf3 and anempty control vector (Ctrl, N=6), with a mixture of Csf3 and humanDNase1 (hDnase1, N=12), and with Csf3 and murine DNase1 (mDnase1, N=5).P-values were calculated using log-rank test.

FIG. 22A-FIG. 22B shows the characterization of rodent-like humanDNase1-variants in vivo and in vitro. (FIG. 20A) Gene therapy withrodent-like DNase1-variants provides full protection from lethalvascular occlusion by NETs. Chronic neutrophilia with concomitantintravascular NET-formation was induced as by hepatic expression ofCsf3, which encodes with G-CSF. The data show the survival curve ofCsf3-expressing Dnase1^(−/−)Dnase1l3^(−/−) mice. Animals were injectedwith Csf3 and an empty control vector (Ctrl, N =6), with a mixture ofCsf3 and human DNase1 (hDnase1, N=12) as shown in FIG. 21. A third groupof Dnase1^(−/−)Dnase1l3^(−/−) mice received an injection with Csf3 andthe gene encoding with murine-like DNase1-variant, which contains 4amino acid mutations Q31R/Q49K/Q79R/W209R (Q31R/Q49K/Q79R/W209R, N=6). Afourth group of Dnase1^(−/−)Dnase1l3^(−/−) mice received an injectionwith Csf3 and the gene encoding with rat-like DNase1-variant, whichcontains 6 amino acid mutations Q31R, Q49K, Q79R, Q177K, W209R, andG262K (Q31R/Q49K/Q79R/Q177K/W209R/G262K, N=6). P-values were calculatedusing log-rank test. (FIG. 22B) Zymography showed dsDNA degradingactivity as dark circles. The dsDNA degrading activity correlates withthe diameter. Samples without activity show the loading well as smallblack spot (e.g. Ctrl). Agarose gel electrophoresis (AGE) of DNAisolated from digested chromatin shows a shift from high-molecularweight DNA to lower or low-molecular weight DNA that correlates withchromatin degrading activity. The murine-like variant of human DNase1(Q31R/Q49K/Q79R/W209R), and the rat-like variant of human DNase1(Q31R/Q49K/Q79R/Q177K/W209R/G262K) show similar dsDNA but more chromatindegrading activity than wild-type human DNase1.

FIG. 23 shows the amino acid sequence alignment of human DNase1 (D1, SEQID NO: 1) and human DNase1L3 (D1L3, SEQ ID NO: 2). Sequence rulersdemark the amino acid positions. Amino acids of D1 with publishedmutations are highlighted. Human D1L3 contains three mutations that areassociated with a gain-of-function in human DNase1 (Q31R/T227K/A136F)and four mutations that are not linked to increased activity.

FIG. 24A-24B show the characterization of DNase1 variants (D1^(v)) withthe mutations Q31R, T227K, and/or A136F. (FIG. 24A) Culture supernatantsof transfected HEK293 cells were analyzed. HEK cells transfected withwild type DNase1 (D1) and DNase1L3 (D1L3) served as controls. Zymographyshowed dsDNA degrading activity as dark circles. The dsDNA degradingactivity correlates with the diameter. Samples without activity show theloading well as small black spot (e.g. Ctrl). Agarose gelelectrophoresis (AGE) of DNA isolated from digested chromatin shows ashift from high-molecular weight DNA to lower or low-molecular weightDNA that correlates with chromatin degrading activity. The D1^(v) thatfeatures three amino acid mutations Q31R/T227K/A136F has similar dsDNAdegrading activity, but a greater capacity to degrade chromatin than D1and D1^(v) featuring Q31R, T227K, A136F, or Q31R/T227K. (FIG. 24B) Genetherapy with a hyperactive D1^(v) provides full partial protection fromlethal vascular occlusion by NETs. Chronic neutrophilia with concomitantintravascular NET-formation was induced as by hepatic expression ofCsf3, which encodes with G-CSF. The data show the survival curve ofCsf3-expressing Dnase1^(−/−)Dnase1l3^(−/−) mice. Animals were injectedwith Csf3 and an empty control vector (Ctrl, N=6), with a mixture ofCsf3 and human DNase1 (hDnase1, N=12) as shown in FIG. 21. In addition,a group of Dnase1^(−/−)Dnase1l3^(−/−) mice received an injection withCsf3 and the gene encoding with a human DNase1-variant, which containsthe triple amino acid mutation Q31R/T227K/A136F (Q31R/T227K/A136F, N=6). P-values were calculated using log-rank test.

FIG. 25 shows a comparison of wild-type DNase1 (D1) and theDNase1-variants (D1^(v)) featuring the mutations Q31R/T227K/A136F.Purified proteins were analyzed at indicated amounts. Zymography showeddsDNA degrading activity as dark circles. The dsDNA degrading activitycorrelates with the diameter. Samples without activity show the loadingwell as small black spot (e.g. 0 ng). Agarose gel electrophoresis (AGE)of DNA isolated from digested chromatin shows a shift fromhigh-molecular weight DNA to lower or low-molecular weight DNA thatcorrelates with chromatin degrading activity. The DNase1-variantfeaturing the mutations Q31R/T227K/A136F degrades chromatinapproximately 10-20-fold more efficiently than wild-type DNase1.

FIG. 26 shows a comparison of DNase1 variants (D1^(v)) with the aminoacid mutations A164K, A136F, and A164K/A136F with wild-type DNase1 (D1)and wild-type (D1L3). Culture supernatants of transfected HEK cells wereanalyzed. Zymography showed dsDNA degrading activity as dark circles.The dsDNA degrading activity correlates with the diameter. Sampleswithout activity show the loading well as small black spot (e.g. Ctrl).Agarose gel electrophoresis (AGE) of DNA isolated from digestedchromatin shows a shift from high-molecular weight DNA to lower orlow-molecular weight DNA that correlates with chromatin degradingactivity. DNase1-variants featuring A136F/A164K degrades chromatin mostefficiently.

FIG. 27A-FIG. 27B shows development of a DNase1 variant with themutation A226_T227insK. (FIG. 26A) Amino acid sequence alignment ofhuman DNase1 (SEQ ID NO: 1) and human DNase1L3 (SEQ ID NO: 2). Sequencerulers demark the amino acid positions. Critical arginine residues inDNase1L3 are highlighted. Shared amino acids between DNase1 and DNase1L3are highlighted and serve as anchors to replace the amino acids sequenceATP of DNase1 with VKKS of DNase1L3. (FIG. 26B) Zymography showed dsDNAdegrading activity as dark circles. The dsDNA degrading activitycorrelates with the diameter. Samples without activity show the loadingwell as small black spot (e.g. Ctrl). The gel was loaded with wild-typeDNase1 (D1), DNase1 variants (D1^(v)) that feature the mutationsA226_T227insK, T227K, and A226_P228delinsVKKS. A226_T227insK, but notA226_P228delinsVKKS, is associated with a reduced dsDNA degradingactivity.

FIG. 28 shows the structure of human DNase1 [4AWN]. Highlighted are theN-terminal beta-sheet and the C-terminal beta-sheet and the motiveS272/D273/H274, which is conserved among D1-protein family members.

FIG. 29 shows the concept of building block engineering of homologousproteins. The technology transfers single or multiple variable aminoacids, which are flanked by conserved single or multiple variable aminoacids, between a donor and recipient protein.

FIG. 30 shows an amino acid sequence alignment of DNase1 and DNase1L3 ofmouse (SEQ ID NOS: 3 and 34), rat (SEQ ID NOS: 4 and 35), chimpanzee(SEQ ID NOS: 36 and 37), and human (SEQ ID NOS: 1 and 2). The N-terminalsignal peptide and conserved amino acids are highlighted. Variable aminoacids are not highlighted and serve as Building Blocks that can betransferred from DNase1 to DNase1L3 and vice versa. Abbreviations: AA,amino acid.

FIG. 31A-FIG. 31B show lists of Building Blocks in human DNase1 (D1) andhuman DNase1L3 (D1L3). FIG. 31A shows amino acids that are conserved inD1 and D1L3, which serve as N- and C-anchors, respectively. Buildingblocks are variable amino acids in D1 and D1L3. Mutations that transferBuilding Blocks from D1L3 to D1 are shown. FIG. 31B shows N- andC-anchors in D1L3. Mutations that transfer Building Blocks from D1 toD1L3 are listed. AA: amino acid.

FIG. 32 shows an application of the building block engineering ofhomologous proteins. The application uses as an initial screening step,the transfer of clusters of building blocks between a homologous donorand recipient protein. Additional optional steps are the transfer ofindividual building blocks, followed by the transfer of individual aminoacids. In a final step (not shown), multiple amino acids, buildingblocks, and building block clusters may be combined to degenerate achimeric enzyme.

FIG. 33 shows characterization of DNase1 variants (D1^(v)) featuringbuilding blocks from DNase1L3 (D1L3). Zymography showed dsDNA degradingactivity as dark circles. The dsDNA degrading activity correlates withthe diameter. Samples without activity show the loading well as smallblack spot (e.g. Ctrl). Agarose gel electrophoresis (AGE) of DNAisolated from digested chromatin shows a shift from high-molecularweight DNA to lower or low-molecular weight DNA that correlates withchromatin degrading activity. Building block substitutions that cause anincrease in chromatin degrading activity are highlighted in dark shade.Samples without such effect are shown in light shade. A DNase1 variantfeaturing the combination of building blocks 11, 12-14, 26, 41-48, and49 shows similar chromatin degrading activity than wild-type DNase1L3.

FIG. 34A-FIG. 34B shows the development of a DNase1L3 variant withoutthe C-terminal tail of wild-type DNase1L3. FIG. 34A shows an amino acidsequence alignment of human DNase1 (SEQ ID NO: 1) and human DNase1L3(SEQ ID NO: 2). Sequence rulers demark the amino acid positions. TheC-terminal tail of DNase1L3 is highlighted and its nuclear localizationsignal is boxed. Shared amino acids between DNase1 and DNase1L3 areindicated. Amino acids located after the conserved SDH motive wereexchanged the building block cluster 60-62 between DNase1 and DNase1L3.FIG. 34B shows zymography showing dsDNA degrading activity as darkcircles. The dsDNA degrading activity correlates with the diameter.Samples without activity show the loading well as small black spot (e.g.Ctrl). Agarose gel electrophoresis (AGE) of DNA isolated from digestedchromatin shows a shift from high-molecular weight DNA to lower orlow-molecular weight DNA that correlates with chromatin degradingactivity. The gels were loaded with wild-type DNase1 (D1), DNase1variant (D1^(v)) that features the BB cluster 60-62 from D1, DNase1L3variant (D1L3^(v)) that features the BB cluster 60-62 from D1, and aD1L3^(v) with two mutations (K301A, K303A) were made to inactivate theNLS2. D1L3^(v) with BB cluster 60-62 from D1 showed increased chromatindegrading activity compared to wild-type D1L3.

FIG. 35A-FIG. 35B shows the development of glycosylated DNase1L3variants (D1L3^(v)). FIG. 35A shows the amino acid sequence alignment ofhuman DNase1 (SEQ ID NO: 1) and human DNase1L3 (SEQ ID NO: 2). Sequencerulers demark the amino acid positions. The glycosylation motivesN40-X-T42 and N128-X-T130 of DNase1 are highlighted. The sites are partof building block (BB) 4 and building block cluster (BB) 23-25. Relevantshared amino acids between DNase1 and DNase1L3 are highlighted. (FIG.35B) Zymography showed dsDNA degrading activity as dark circles. ThedsDNA degrading activity correlates with the diameter. Samples withoutactivity show the loading well as small black spot (e.g. Ctrl). Agarosegel electrophoresis (AGE) of DNA isolated from digested chromatin showsa shift from high-molecular weight DNA to lower or low-molecular weightDNA that correlates with chromatin degrading activity. The gels wereloaded with wild-type DNase1L3 (D1L3), DNase1L3 variant (D1L3^(v)) thatfeatures BB 4 from D1, and DNase1L3 variant (D1L3^(v)) th_(a)t featuresthe BB cluster 23-25 from D1. A Western Blot (WB) with antibodiesagainst D1L3 detected that D1L3^(v) showed increased amounts of largerproteins. Both glycosylated DNase1L3 variants (D1L3^(v)) retained theircapacity to degrade chromatin.

FIG. 36A-FIG. 36F shows the therapeutic effect of DNase1L3 (SEQ ID NO:2) and a variant thereof (SEQ ID NO: 17) against NETs in vivo. FIG. 36Ashows an H&E staining of a mouse heart with a hematoxylin-rich and denseclot of NETs within a cardiac chamber. FIG. 36B shows a time linestarting with the induction of NETs, leading to vascular occlusion byNETs and death. Hematuria and hypothermia are external signs for in vivoNET-clots. (FIG. 36C) Outline of the experimental design: Csf3 wasexpressed in DNase1^(−/−)Dnase1l3^(−/−) to induce vascular clots ofNETs. Mice that showed hematuria and hypothermia were randomized by aninjection of vehicle (N=3), dornase alpha (D1, N=3), purified DNase1L3(D1L3, N=3), or a purified DNase1L3 variant (SEQ ID NO: 17, D1L3^(v),N=3). After 30 minutes lungs and serum were collected. The therapy withD1L3 or D1L3^(v), but not saline or D1, significantly reduced vascularocclusion by NETs in lungs (FIG. 36D) and increased the levels of DNA inserum (FIG. 36E). FIG. 36F shows oligo-nucleosomes in the serum frommice receiving the D1L3 therapy. Mice receiving D1L3^(v) showed smallermono- and di-nucleosomes. DNA isolates from serum of mice receiving D1therapy showed faint DNA smears of various sizes, whereas no DNA couldbe isolated from serum of mice injected with vehicle.

FIG. 37A-FIG. 37C shows the therapeutic effect of a DNase1L3 variant(SEQ ID NO: 17) in a rat model of ischemia-reperfusion (IR) injury.(FIG. 37A) Outline of the experimental design: One testicle of malewild-type rats was subjected to testicular torsion. The procedure causesan ischemic tissue injury. Mice were randomized. A de-torsion of thetesticle was performed, followed by an injection of vehicle (N=6),dornase alpha (D1, N=6), or a purified DNase1L3 variant (SEQ ID NO: 17,D1L3^(v), N=6). The untreated and the ischemic testicle were collectedafter 7 days. FIG. 37B shows that the therapy with the D1L3^(v), but notsaline or D1, significantly reduced atrophy in the ischemic testicle.FIG. 37C quantified the macroscopic tissue injury. All untreatedtesticles are brownish and vascularized, indicating healthy tissue(Ctrl, Score: 0). Testicular torsion followed by reperfusion damagedtissue, which appeared whitish (IR, Score: 4). Therapy with theD1L3^(v), but not saline or D1, substantially reduced the tissue injuryand 4/6 testicles showed no signs of injury. Scale bar: 1 cm.

FIG. 38A-FIG. 38C shows the expression levels of DNase1, DNase1L3, andvariants thereof (FIG. 38A) Stable pools of CHO cells producingwild-type DNase1 (D1), a variant thereof (D1^(v), SEQ ID NO: 7),wild-type D1L3, and a variant thereof (D1L3^(v), SEQ ID NO: 17) werecultured in bioreactors. Samples were collected periodically andanalyzed by Western Blot. Expression of D1 and D1^(v) was detected, butonly low levels of D1L3 and D1L3^(v). (FIG. 38B) Robust expression ofD1L3 and D1L3^(v)was achieved in Pichia pastoris. (FIG. 38C) Zymographyshowed dsDNA degrading activity as dark circles. The dsDNA degradingactivity correlates with the diameter. Samples without activity show theloading well as small black spot (e.g. D1L3, 0.0005 ng). Agarose gelelectrophoresis (AGE) of DNA isolated from digested chromatin shows ashift from high-molecular weight DNA to lower or low-molecular weightDNA that correlates with chromatin degrading activity. D1L3^(v)degradeschromatin approximately 10-fold more efficiently than D1L3.

FIG. 39 illustrates certain DNase variants in accordance with thisdisclosure. The amino acid mutations in DNase1 and DNase1L3 with signalpeptide as well as the mature proteins without signal peptide arelisted. Mutations that were generated using Building Block Engineeringare marked with “+” and the Building Blocks are indicated in brackets.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides engineered DNase proteins, members of theDNase1-protein family (including DNase1-like 3 and DNase1) that areuseful for treating conditions characterized by neutrophil extracellulartrap (NET) accumulation and/or release. In some aspects, the inventionprovides compositions and methods for preventing or treating vascularocclusion involving NETs. As demonstrated herein, NETs participate in anon-canonical mechanism for vascular occlusion, which is not dependenton fibrin and platelets.

As used herein, the term “neutrophil extracellular trap” or “NET” refersto any extracellular trap (“ET”) comprising extracellular DNA formed bycells such as, but not limited to, neutrophils, monocytes, macrophages,basophils, eosinophils, mast cells, cancer cells, injured cells (e.g.,injured endothelial cells), and the like. Unless the context indicatesotherwise, the terms NET and ET are used interchangeably herein.

There are two extracellular DNase enzymes found in the circulation ofhumans and mice, DNase1 (D1) and DNase1-like 3 (D1L3). These enzymes canbe deficient in their ability and mechanism to degrade NETs in vitro andin vivo, and are found at relatively low concentrations in humans andmice. The natural enzymes do not have ideal physical, enzymatic, and/orpharmacodynamic properties for recombinant therapy, and particularly forsystemic therapy.

D1 and D1L3 belong, along with DNase1 -like 1 (D1L1) and DNase1-like 2(D1L2), to the DNase1-protein family. D1 and D1L3 are expressed in avariety of species including, humans, primates, and rodents. In humansand mice, D1 and D1L3 show a protein similarity of 49-52%. However,despite their homology, D1 and D1L3 differ in cellular origin,sensitivity towards inhibitors, and substrate affinity. D1preferentially cleaves protein-free DNA (e.g. bacterial DNA, plasmidDNA), whereas D1L3 targets chromatin, the complex of DNA and histones,which is commonly found in the nucleus of eukaryotic cells. D1 activityis inhibited upon binding to monomeric actin and sensitive tophysiological salt concentrations. In addition, D1 is glycosylated atN40 (corresponds to N18 in the mature enzyme without signal peptide) andN128 (N106), which makes the enzyme resistant to inactivation by serumproteases. In contrast, D1L3 lacks glycosylation and actin-bindingsites, which causes its susceptibility towards several proteases andresistance towards actin, respectively.

In some aspects, the invention provides D1L3 variants engineered to havephysical, pharmacodynamic, and/or enzymatic properties more suitable fortherapy, for example, to reduce or prevent NET accumulation in asubject. In various embodiments, the invention provides a recombinantD1L3 protein variant comprising: one or more glycosylations, one or moreamino acid alterations resulting in increased substrate affinity,inactivation of a nuclear localization signal, deletion of all or partof a C-terminal tail, pegylation, and fusion to a carrier protein ormoiety. In various embodiments, the D1L3 variant has increased proteinstability, increased protease-resistance, increased bioavailability,increased half-life in circulation, and/or substantially the same orbetter DNA and/or chromatin and/or NET-degrading activity, higherproduction levels with in vitro expression systems (e.g. Chinese hamsterovary cells and/or Pichia pastoris) as compared to wild-type D1L3protein of SEQ ID NO:2.

In various embodiments, the D1L3 variant comprises an amino acidsequence with at least 80% sequence identity to the enzyme defined bythe amino acid sequence of SEQ ID NO:2, with one or more amino acidmodifications with respect to SEQ ID NO:2 as described herein. In someembodiments, D1L3 variant comprises an amino acid sequence with at least90%, or at least 95%, or at least 97%, or at least 98% sequence identitywith with the DNase enzyme defined by SEQ ID NO:2.

As used herein, when referring to sequence identity with wild-type DNaseenzymes, and unless stated otherwise, sequences refer to mature enzymeslacking the signal peptide. Further, unless stated otherwise, amino acidpositions are numbered with respect to the full translated DNasesequence, including signal peptide, for clarity. Accordingly, forexample, reference to sequence identity to the enzyme of SEQ ID NO:2refers to a percent identity with the mature enzyme having M21 at theN-terminus. Similarly, reference to sequence identity to the enzyme ofSEQ ID NO:1 (human D1) refers to a percent identity with the matureenzyme having L23 at the N-terminus.

The similarity of nucleotide and amino acid sequences, i.e. thepercentage of sequence identity, can be determined via sequencealignments as known in the art. Such alignments can be carried out withseveral art-known algorithms, such as with the mathematical algorithm ofKarlin and Altschul (Karlin & Altschul (1993) Proc. Natl. Acad. Sci. USA90: 5873-5877), with hmmalign (HMMER package, http://hmmer.wustl.edu/)or with the CLUSTAL algorithm (Thompson, J. D., Higgins, D. G. & Gibson,T. J. (1994) Nucleic Acids Res. 22, 4673-80). Exemplary algorithms areincorporated into the BLASTN and BLASTP programs of Altschul et al(1990) J. Mol. Biol. 215: 403-410. When utilizing BLAST programs, thedefault parameters of the respective programs are used.

In some embodiments, the recombinant D1L3 protein variant comprises oneor more glycosylation consensus sequences, e.g., NX(T/S). The human wildtype enzyme (SEQ ID NO:2) does not contain a consensus glycosylationsequence. However, engineering a glycosylation consensus sequence intoD1L3 can provide for glycosylation (and protease resistance) withoutsubstantial impact on the enzyme activity. In some embodiments, theglycosylation consensus sequence includes a D38N substitution and anN4OT or N40S substitution, and/or an A127N substitution and a V129T orV129S substitution from the wild type sequence (e.g., SEQ ID NO:2). Insome embodiments, the recombinant D1L3 enzyme has one, two, three, orfour glycosylation sites. The consensus sequence can be introduced intothe protein at a location that does not affect its enzymatic activity,and in some embodiments is located near portions of the enzyme that areimportant for maintaining structure of the enzyme (e.g., such as withinamino acids 25 to 289, and in some embodiments, within amino acids 24 to150).

In some embodiments, the invention provides a DNase enzyme comprising anamino acid sequence that is at least 80% identical to the amino acidsequence of the enzyme defined by SEQ ID NO:2, wherein the enzyme has asubstitution of asparagine at the position corresponding to position 38and/or position 127 of SEQ ID NO:2, and the N38 and/or N127 isglycosylated. In some embodiments, the substitution at position 38and/or 127 is made via a building block substitution from SEQ ID NO:1 asdescribed herein.

In various embodiments, D1L3 variants having one or more glycosylationsexhibit increased resistance to serum protease degradation and/or anincreased serum half-life compared to wild-type D1L3 protein (SEQ IDNO:2).

In some embodiments, D1L3 protein variant comprises an inactivation of anuclear localization signal (NLS). D1L3 features two nuclearlocalization sites (NLS1, NLS2), which both target the enzyme to thenucleus during apoptosis. Indeed, D1L3 is required for fragmentation ofnuclear DNA in apoptotic and necrotic cells in vivo. NLS1 is locatednear the N-terminus (about amino acid positions 80 to 96 with respect toSEQ ID NO: 2). NLS2 (amino acid positions 291 to 304 with respect to SEQID NO: 2) is embedded within a C-terminal tail (amino acid positions 283to 305 with respect to SEQ ID NO: 2) that is unique to D1L3 and notpresent in D1. The C-terminal tail is thought to be required for D1L3 todegrade different substrates of extracellular DNA, namelylipid-encapsulated DNA and chromatin within apoptotic bodies. Lipidsencapsulate DNA during transfections. In brief, cDNA, protein-free DNA,and cationic lipids form a complex that penetrates through the plasmamembrane of target cells. D1L3 interferes with transfections and theC-terminal tail is critical for this function. Physiological substratesof D1L3 are apoptotic bodies, extracellular lipid vesicles filled withchromatin from apoptotic cells. The C-terminal tail enables D1L3 topenetrate through lipid membranes of apoptotic bodies and degrade thechromatin load. The C-terminal tail is also thought to be required fordegradation of lipid-free, extracellular chromatin by D1L3. Thus, theC-terminal tail, a molecular feature unique to D1L3, appears to beresponsible for the distinct substrate affinities of D1 and D1L3.

Inactivation of one or more NLSs enhances the availability and/oractivity of the enzyme in the circulation. In some embodiments, the NLSinactivation is by deletion of all or part of NLS1 and/or NLS2. In someembodiments, the NLS is inactivated by substitution and/or deletion ofamino acids within NLS1 and/or NLS2. In some embodiments, NLS2 isdeleted, entirely or partially. In some embodiments, the D1L3 proteinvariant comprises a deletion of all or part of the C-terminal tail,and/or substitution of one or more amino acids in the C-terminal tail.

In certain embodiments, the D1L3 variant contains one or more, e.g., 1,2, 3, 4, 5, or more amino acid substitutions, additions (e.g.,insertions), or deletions in the NLS1 domain. The NLS2 domain or aportion thereof may be deleted. In certain embodiments, the D1L3 proteinvariant contains one or more, e.g., 1, 2, 3, 4, 5, or more amino acidsubstitutions, additions, or deletions in the C-terminal tail domain.The C-terminal tail domain or a portion thereof may be deleted. Invarious embodiments, the D1L3 protein variant contains one or more,e.g., 1, 2, 3, 4, 5, or more amino acid substitutions, additions, ordeletions in the NLS2 domain which is located in the C-terminal taildomain. The NLS2 domain or a portion thereof can be deleted in the D1L3protein variant.

In various aspects, the invention provides a protein engineeringtechnology that is based on a transfer of a single amino acid ormultiple-adjacent amino acids, termed “building block”, between twomembers of a protein family, such as DNase1-protein family members togenerate enzymatically active variants of DNase1-protein family members(including variant of D1 and D1L3). A “building block” is defined byamino acids that are variable between two or more members of the DNase1-protein family. These variable amino acids are flanked by amino acidsthat are conserved between two or more members of the DNase1 -proteinfamily (“anchors”). The variable single amino acid or multiplecontiguous amino acids (“building blocks”) are exchanged between membersof the DNase1-protein family by implanting them between conserved singleamino acid or multiple contiguous amino acids (“anchors”).

This approach is referred to herein as “building-block proteinengineering.” Where three or more amino acids are transferred, up to ⅓of the amino acids may be further substituted. For example, where wherethree or six amino acids are transferred as a building block, one or upto two resides may be further substituted, respectively. In someembodiments, four or more amino acids are transferred as a buildingblock substitution, and up to 25% of the transferred amino acids arefurther substituted, e.g., with conservative or non-conservative aminoacid modifications. For example, where four, eight, or twelve aminoacids are transferred, one, two, or three amino acids (respectively) maybe further substituted in the building block substitution.

In various aspects (including in connection with embodiments describedabove), the D1L3 variant comprises at least one building blocksubstitution from D1. FIG. 31 illustrates 62 building blocksubstitutions between D1 and D1L3.

Exemplary building block substitutions from D1 include one or more of:

substitution of 1-20 (MSRELAPLLLLLLSIHSALA) of SEQ ID NO:2 with 1-22(MRGMKLLGALLALAALLQGAVS) from SEQ ID NO:1; substitution of 21-25 (MRICS)of SEQ ID NO:2 with 23-27 (LKIAA) from SEQ ID NO:1; substitution of28-30 (VRS) of SEQ ID NO:2 with 30-32 (IQT) from SEQ ID NO:1;substitution of 33-34 (ES) of SEQ ID NO:2 with 35-36 (ET) from SEQ IDNO:1; substitution of 36-45 (QEDKNAMDVI) of SEQ ID NO:2 with 38-47(MSNATLVSYI) from SEQ ID NO:1; substitution of 47-51 (KVIK) of SEQ IDNO:2 with 49-53 (QILS) from SEQ ID NO:1; substitution of 52 (C) of SEQID NO:2 with 54 (Y) from SEQ ID NO:1; substitution of 54-58 (IILVM) ofSEQ ID NO:2 with 56-60 (IALVQ) from SEQ ID NO:1; substitution of 60-61(IK) of SEQ ID NO:2 with 62-63 (VR) from SEQ ID NO:1; substitution of63-70 (SNNRICPI) of SEQ ID NO:2 with 65-72 (SHLTAVGK) from SEQ ID NO:1;substitution of 72-74 (MEK) of SEQ ID NO:2 with 74-76 (LDN) from SEQ IDNO:1;

substitution of 77-84 (RNSRRGIT) of SEQ ID NO:2 with 79-84 (QDAPDT) fromSEQ ID NO:1; substitution of 86 (N) of SEQ ID NO:2 with 86 (H) from SEQID NO:1; substitution of 88-89 (VI) of SEQ ID NO:2 with 88-89 (VV) fromSEQ ID NO:1;

substitution of 91-92 (SR) of SEQ ID NO:2 with 91-92 (EP) from SEQ IDNO:1; substitution of 96-97 (NT) of SEQ ID NO:2 with 96-97 (NS) from SEQID NO:1; substitution of 101 (Q) of SEQ ID NO:2 with 101 (R) from SEQ IDNO:1; substitution of 103 (A) of SEQ ID NO:2 with 103 (L) from SEQ IDNO:1; substitution of 105 (L) of SEQ ID NO:2 with 105 (V) from SEQ IDNO:1; substitution of 107-110 (KEKL) of SEQ ID NO:2 with 107-110 (RPDQ)from SEQ ID NO:1; substitution of 113-116 (VKRS) of SEQ ID NO:2 with113-116 (AVDS) from SEQ ID NO:1; substitution of 118 (H) of SEQ ID NO:2with 118 (Y) from SEQ ID NO:1; substitution of 120 (H) of SEQ ID NO:2with 120 (D) from SEQ ID NO:1; substitution of 122-127 (YQDGDA) of SEQID NO:2 with 122-128 (GCEPCGN) from SEQ ID NO:1; substitution of 129 (V)of SEQ ID NO:2 with 130 (T) from SEQ ID NO:1; substitution of 131 (S) ofSEQ ID NO:2 with 132 (N) from SEQ ID NO:1; substitution of 135-136 (FV)of SEQ ID NO:2 with 136-137 (AIV) from SEQ ID NO:1; substitution of 138(W) of SEQ ID NO:2 with 139 (R) from SEQ ID NO:1; substitution of140-143 (QSPH) of SEQ ID NO:2 with 141-144 (FSRF) from SEQ ID NO:1;substitution of 145-148 (AVKD) of SEQ ID NO:2 with 146-149 (EVRE) fromSEQ ID NO:1; substitution of 150 (V) of SEQ ID NO:2 with 151 (A) fromSEQ ID NO:1; substitution of 152 (I) of SEQ ID NO:2 with 153 (V) fromSEQ ID NO:1; substitution of 156-157 (TT) of SEQ ID NO:2 with 157-158(AA) from SEQ ID NO:1; substitution of 159-161 (ETS) of SEQ ID NO:2 with160-162 (GDA) from SEQ ID NO:1; substitution of 163 (K) of SEQ ID NO:2with 164 (A) from SEQ ID NO:1; substitution of 167 (E) of SEQ ID NO:2with 168 (A) from SEQ ID NO:1; substitution of 169-170 (VE) of SEQ IDNO:2 with 170-171 (YD) from SEQ ID NO:1; substitution of 173 (T) of SEQID NO:2 with 174 (L) from SEQ ID NO:1; substitution of 176-178 (KHR) ofSEQ ID NO:2 with 177-179 (QEK) from SEQ ID NO:1; substitution of 180-181(KA) of SEQ ID NO:2 with 181-182 (GL) from SEQ ID NO:1; substitution of183-186 (NFIF) of SEQ ID NO:2 with 184-187 (DVML) from SEQ ID NO:1;substitution of 198-201 (PKKA) of SEQ ID NO:2 with 199-202 (RPSQ) fromSEQ ID NO:1; substitution of 203-204 (KN) of SEQ ID NO:2 with 204-205(SS) from SEQ ID NO:1; substitution of 208 (R) of SEQ ID NO:2 with 209(W) from SEQ ID NO:1; substitution of 210 (D) of SEQ ID NO:2 with 211(S) from SEQ ID NO:1; substitution of 212 (R) of SEQ ID NO:2 with 213(T) from SEQ ID NO:1; substitution of 214 (V) of SEQ ID NO:2 with 215(Q) from SEQ ID NO:1; substitution of 218 (G) of SEQ ID NO:2 with 219(P) from SEQ ID NO:1; substitution of 220-221 (QE) of SEQ ID NO:2 with221-222 (SA) from SEQ ID NO:1; substitution of 225-228 (VKKS) of SEQ IDNO:2 with 226-228 (ATP) from SEQ ID NO:1; substitution of 230 (N) of SEQID NO:2 with 230 (H) from SEQ ID NO:1; substitution of 238-240 (LRG) ofSEQ ID NO:2 with 238-240 (VAG) from SEQ ID NO:1; substitution of 241-246(QEIVSS) of SEQ ID NO:2 with 241-246 (MLLRGA) from SEQ ID NO:1;substitution of 250 (K) of SEQ ID NO:2 with 250 (D) from SEQ ID NO:1;substitution of 252-254 (NSV) of SEQ ID NO:2 with 252-254 (ALP) from SEQID NO:1; substitution of 256 (D) of SEQ ID NO:2 with 256 (N) from SEQ IDNO:1; substitution of 259-260 (KA) of SEQ ID NO:2 with 259-260 (AA) fromSEQ ID NO:1; substitution of 262 (K) of SEQ ID NO:2 with 262 (G) fromSEQ ID NO:1; substitution of 264-267 (TEEE) of SEQ ID NO:2 with 264-267(SDQL) from SEQ ID NO:1; substitution of 269-271 (LDV) of SEQ ID NO:2with 269-271 (QAI) from SEQ ID NO:1; substitution of 275 (F) from SEQ IDNO:2 with 275 (Y) from SEQ ID NO:1; substitution of 279-280 (FK) of SEQID NO:2 with 279-280 (VM) from SEQ ID NO:1; and

substitution of 282-305 (QSSRAFTNSKKSVTLRKKTKSKRS) of SEQ ID NO:2 with282 (K) from SEQ ID NO:1.

These building block substitutions from D1 result in variants of D1L3which feature one or more of the following mutations:M1_A20delinsMRGMKLLGALLALAALLQGAVS, M21_S25delinsLKIAA, V28_S30delinsIQT, E33_S34delinsET, Q36_I45delinsMSNATLVSYI,K47_K50delinsQILS, C52Y, 154_M58delinsIALVQ, 160_K61delinsVR,S63P_I70delinsSHLTAVGK, M72_K74delinsLDN, R77_T84delinsQDAPDT, N86H,V88_I89delinsVV, S91_R92delinsEP, N96_T97delinsNS, Q101R, A103L, L105V,K107P_L110delinsRPDQ, V113_S116delinsAVDS, H118Y, H120D,Y122_A127delinsGCEPCGN, V129T, S131N, 135F_136VdelinsAI, W138R,Q140_H143delinsFSRF, A145_D148delinsAVKD, V150A, I152A,T156_T157delinsAA, E159_S161delinsGDA, K163A, E167A, V169 E170delinsYD,T173L, K176_R178delinsQEK, K180_A181delinsGL, N183_F186delinsDVML,P198_A201delinsRPSQ, K203_N204delinsSS, R208W, D210S, R212T, V214Q,G218P, Q220_E221delinsSA, V225_S228delinsATP, N230H, L238_R239delinsVA,Q241_S246delinsMLLRGA, K250D, N252_V254delinsALP, D256N,K259_A260delinsAA, K262G, T264_E267delinsSDQL, L269_V271delinsQAI,F275Y, F279_K280delinsVM, Q282_S305delinsK.

The term “delins” refers to a deletion between and including twoindicated amino acids, with an insertion of an amino acid or sequence ofamino acids at the site of the deletion. For example, the notationE91_P92delinsSR means that the amino acids from E91 to P92 are deletedand the amino acids SR are inserted at the site of the deletion (e.g.,the resulting amino acid sequence will have S91 and R92).

The term “ins” refers to an insertion of amino acids between twoindicated amino acids. For example, the notation E91_P92insSR means thatthe amino acids SR are inserted between E91 and P92, resulting in thesequence E91, S92, R93, and P94.

Multiple mutations in one enyzme are separared by “/” e.g., T227K/A136ForR199_Q202delinsPKKA/S204_S205delinsKN/W209R/S211D/T213R/Q215V/P219G/S221_A222delinsQE.

In some embodiments, the D1L3 variant comprises one or more adjacentbuilding block substitutions. For example, the D1L3 variant may comprisewith respect to SEQ ID NO:2 the mutationF275Y/F279_K280delinsVM/Q282_S305delinsK (SEQ ID NO: 17). In someembodiments, the D1L3 variant may comprise with respect to SEQ ID NO:2the mutation Q36_I45delinsMSNATLVSYI (SEQ ID NO: 19). In someembodiments, the D1L3 variant may comprise with respect to SEQ ID NO:2the mutation Y122_A127delinsGCEPCGNN129T/S131N (SEQ ID NO: 20).

In an exemplary embodiment, the D1L3 variant comprises 2 or 3substitutions selected from the mutations with respect to SEQ ID NO:2:Q36_I45delinsMSNATLVSYI, Y122_A127delinsGCEPCGNN129T/S131N,F275Y/F279_K280delinsVM/Q282_S305delinsK.

Thus, in some embodiments, the invention provides a DNase enzymecomprising an amino acid sequence that is at least 80% identical to theamino acid sequence of the enzyme defined by SEQ ID NO:2, and comprisesat least one building block substitution from the amino acid sequence ofSEQ ID NO:1. Building block substitutions between SEQ ID NOS: 1 and 2are illustrated and numbered in FIG. 31. For example, in someembodiments, the enzyme comprises the mutationF275Y/F279_K280delinsVM/Q282_ S305delinsK, and which optionallycomprises the amino acid sequence of SEQ ID NO: 17. In some embodiments,the enzyme comprises the mutations Q36_I45delinsMSNATLVSYI,Y122_A127delinsGCEPCGN, V129T, or S131N, and optionally has the aminoacid sequence of SEQ ID NO: 19 or SEQ ID NO: 20.

Exemplary D1L3 variants include those comprising the amino acid sequenceof SEQ ID Nos: 17 to 20. The invention in some embodiments, includesderivatives having from 1 to 10 (e.g., 1 to 5) amino acid insertions,deletions, or substitutions with respect to any one of SEQ ID NOS:17 to20. In some embodiments, the D1L3 variant comprises one or moreadditional block amino acid substitutions from a homologous DNase (e.g.,from D1). Such block substitutions may replace at least 2, at least 3,at least 4, or at least 5 amino acids. In some embodiments, the blocksubstitutions replace from 2 to 20 amino acids, such as from 3 to 15amino acids, or from 3 to 10 amino acids, with the comparable buildingblock from the homologous DNase.

The engineered variants of D1L3 protein may comprise one or moreadditional amino acid substitutions, additions (insertions), deletions,or truncations in the amino acid sequence of human D1L3 (SEQ ID NO: 2).Amino acid substitutions may include conservative and/ornon-conservative substitutions.

For example, “conservative substitutions” may be made, for instance, onthe basis of similarity in polarity, charge, size, solubility,hydrophobicity, hydrophilicity, and/or the amphipathic nature of theamino acid residues involved. The 20 naturally occurring amino acids canbe grouped into the following six standard amino acid groups: (1)hydrophobic: Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser,Thr; Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5)residues that influence chain orientation: Gly, Pro; and (6) aromatic:Trp, Tyr, Phe. “Conservative substitutions” are defined as exchanges ofan amino acid by another amino acid listed within the same group of thesix standard amino acid groups shown above. For example, the exchange ofAsp by Glu retains one negative charge in the so modified polypeptide.In addition, glycine and proline may be substituted for one anotherbased on their ability to disrupt a-helices. As used herein,“non-conservative substitutions” are defined as exchanges of an aminoacid by another amino acid listed in a different group of the sixstandard amino acid groups (1) to (6) shown above.

In some embodiments, the D1L3 protein variant comprises an N-terminal orC-terminal fusion to a half-life extending moiety, such as albumin,transferrin, an Fc, or elastin-like protein. See U.S. Pat. No.9,458,218, which is hereby incorporated by reference in its entirety. Insome embodiments, the D1L3 is dimerized by an immunoglobulin hingeregion. For example, the engineered enzymes described herein may alsoinclude an Fc-fusion domain (e.g. a hinge and CH2 domains and CH3domains of an immunoglobulin). In other cases, the engineered D1L3protein is fused to albumin, e.g., human albumin or a fragment thereof.See WO 2015/066550; U.S. Pat. No. 9,221,896, which are herebyincorporated by reference in its entirety. Albumin can be joined to atthe N-terminus or the C-terminus of the engineered D1 protein, and mayoptionally comprise an amino acid linker. In some embodiments, D1L3 andD1 are together dimerized by an Fc hinge region, creating a dimericmolecule with synergistic functional properties for degrading NETs.

In some embodiments, the recombinant D1L3 protein variant comprises oneor more polyethylene glycol (PEG) moieties, which may be conjugated atone or more of positions or the C-terminus. In some embodiments, thenative amino acid at that position is substituted with an amino acidhaving a side chain suitable for crosslinking with hydrophilic moieties,to facilitate linkage of the hydrophilic moiety to the peptide. In otherembodiments, an amino acid modified to comprise a hydrophilic group isadded to the peptide at the C-terminus. The PEG chain(s) may have amolecular weight in the range of about 500 to about 40,000 Daltons. Insome embodiments, the PEG chain(s) have a molecular weight in the rangeof about 500 to about 5,000 Daltons. In some embodiments, the PEGchain(s) have a molecular weight of about 10,000 to about 20,000Daltons.

In various embodiments, the D1L3 protein variant has increased proteinstability, increased serum availability, and substantially the same orbetter NET-degrading activity (as determined in vitro or in vivo) ascompared to wild-type D1L3 protein. In various embodiments, the D1L3variant comprises one or more of the following properties relative towild type D1L3: higher protein-free DNA (naked DNA) degradationactivity, the same or substantially the same (e.g., at least 50%)chromosomal DNA degradation activity, protease resistance, an increasedhalf-life, and higher production levels with in vitro expression systems(e.g. Chinese hamster ovary cells and/or Pichia pastoris).

DNA fragmentation can be measured using methods known to those skilledin the art such as gel electrophoresis and DNase activity assays.Methods for quantifying NETs, NET degradation, and DNA fragmentationactivity are described in, for example, Hakkim et al., Proc. Natl. Acad.Sci USA, 2010, 107(21):9813-9818; Napirei et al. Biochem J, 2005, 389:355-364. Fuchs et al., J Cell Biol, 2007, 176(2):231-241, Sisirak et al.Cell, 2016, 166:88-101, and Jimenez-Alcazar et al. J Thromb Haemost,2015, 13, 732-742.

In some aspects, the invention provides DNase1 (D1) variants engineeredto have physical, pharmacodynamic, and/or enzymatic properties moresuitable for therapy, for example, to reduce or prevent NET accumulationin a subject. In some embodiments, the variant is more amenable torecombinant expression, thereby providing considerable manufacturingadvantages. In various embodiments, the engineered D1 variant comprisesan amino acid sequence that is at least 80% identical to the matureenzyme defined by the amino acid sequence of SEQ ID NO:1, with one ormore amino acid substitutions, additions, or deletions resulting in oneor more of a mutated DNA binding site, addition of a chromatin bindingsite, a mutated actin binding site, mutation of a glycosylation site,addition of a nuclear localization signal (e.g., having similarity oridentity to NLS1 or NLS2 of D1L3), and/or a C-terminal domain similar tothe C-terminal tail of D1L3. The D1 variant comprises an amino acidsequence with at least 80% identity to the amino acid sequence of theenzyme defined by SEQ ID NO:1, with one or more amino acid modificationswith respect to SEQ ID NO:1 as described herein. In some embodiments,D1L3 variant comprises an amino acid sequence with at least 90%, or atleast 95%, or at least 97%, or at least 98% identity with the enzyme ofSEQ ID NO:1.

In various embodiments, the D1 variant comprises one or more amino acidsubstitutions, additions, deletions, or truncations in the amino acidsequence of human D1 (SEQ ID NO: 1). In some embodiments, the amino acidsubstitutions, and may include conservative and/or non-conservativesubstitutions as described. For example, the D1 variant may have from 1to 20 (or from 1 to 10, or from 1 to 5) amino acid substitutions,deletions, or insertions with respect to the enzyme of SEQ ID NO:1.

In some embodiments, the D1 variant comprises one or more additionalblock amino acid substitutions from a homologous DNase (e.g., fromD1L3). Such block substitutions may each replace from 1 to 24 aminoacids, such as from 3 to 15 amino acids, or from 3 to 10 amino acids,from the homologous DNase. In some embodiments, the D1 variant comprisesfrom 2 to 5 block substitutions from the homologous DNase (e.g., D1L3).In some embodiments 2 or 3 contiguous building block substitutions aretransferred.

In various embodiments, the D1 variant is engineered to comprise one ormore of the following characteristics relative to the wild type enzyme:the substantially the same or higher protein-free DNA (naked DNA)degradation activity (e.g., at least 50% or better), higher chromosomalDNA degradation activity, similar or improved protease resistance, actinresistance, an increased serum half-life, improved penetetration fromblood into urine or bile, or a combination of such properties.

In some embodiments, the engineered D1 enzyme comprises an amino acidsequence comprising at least 50% (or at least 70%, 80%, 90%) sequenceidentity to the C-terminal domain of wild-type D1L3 protein (e.g., aminoacids 283 to 305 of SEQ ID NO:2).

In some embodiments, the D1 enzyme comprises an amino acid sequencehaving at least 50% or at least 75% sequence identity to the NLS1 ofwild-type D1L3 (amino acids 35 to 51 of SEQ ID NO:2), a nuclearlocalization signal 2 (NLS2) having substantial sequence identity to theNLS2 of wild-type D1L3 protein (amino acids 296 to 304 of SEQ ID NO:2).

In various aspects, the recombinant D1 variant comprises one or morebuilding block substitutions selected from: substitution of 1-22(MRGMKLLGALLALAALLQGAVS) from SEQ ID NO:1 with 1-20 (MSRELAPLLLLLLSIHSALA) from SEQ ID NO:2; substitution of 23-27 (LKIAA) from SEQ IDNO:1 with 21-25 (MRICS) from SEQ ID NO:2; substitution of 30-32 (IQT)from SEQ ID NO:1 with 28-30 (VRS) from SEQ ID NO:2; substitution of35-36 (ET) from SEQ ID NO:1 with 33-34 (ES) from SEQ ID NO:2;substitution of 38-47 (MSNATLVSYI) from SEQ ID NO:1 with 36-45(QEDKNAMDVI) from SEQ ID NO:2; substitution of 49-52 (QILS) from SEQ IDNO:1 with 47-50 (KVIK) from SEQ ID NO:2; substitution of 54 (Y) from SEQID NO:1 with 52 (C) from SEQ ID NO:2; substitution of 56-60 (IALVQ) fromSEQ ID NO:1 with 54-58 (IILVM) from SEQ ID NO:2;substitution of 62-63(VR) from SEQ ID NO:1 with 60-61 (IK) from SEQ ID NO:2; substitution of65-72 (SHLTAVGK) from SEQ ID NO:1 with 63-70 (SNNRICPI) from SEQ IDNO:2; substitution of 74-76 (LDN) from SEQ ID NO:1 with 72-74 (MEK) fromSEQ ID NO:2; substitution of 79-84 (QDAPDT) from SEQ ID NO:1 with 77-84(RNSRRGIT) from SEQ ID NO:2; substitution of 86 (H) from SEQ ID NO:1with 86 (N) from SEQ ID NO:2; substitution of 88-89 (VV) from SEQ IDNO:1 with 88-89 (VI) from SEQ ID NO:2; substitution of 91-92 (EP) fromSEQ ID NO:1 with 91-92 (SR) from SEQ ID NO:2; substitution of 96-97 (NS)from SEQ ID NO:1 with 96-97 (NT) from SEQ ID NO:2; substitution of 101(R) from SEQ ID NO:1 with 101 (Q) from SEQ ID NO:2; substitution of 103(L) from SEQ ID NO:1 with 103 (A) from SEQ ID NO:2; substitution of 105(V) from SEQ ID NO:1 with 105 (L) from SEQ ID NO:2; substitution of107-110 (RPDQ) from SEQ ID NO:1 with 107-110 (KEKL) from SEQ ID NO:2;substitution of 113-116 (AVDS) from SEQ ID NO:1 with 113-116 (VKRS) fromSEQ ID NO:2; substitution of 118 (Y) from SEQ ID NO:1 with 118 (H) fromSEQ ID NO:2, substitution of 120 (D) from SEQ ID NO:1 with 120 (H) fromSEQ ID NO:2; substitution of 122-128 (GCEPCGN) from SEQ ID NO:1 with122-127 (YQDGDA) from SEQ ID NO:2, substitution of 130 (TF) from SEQ IDNO:1 with 129 (V) from SEQ ID NO:2; substitution of 132 (N) from SEQ IDNO:1 with 131 (S) from SEQ ID NO:2; substitution of 136-137 (AI) fromSEQ ID NO:1 with 135-136 (FV) from SEQ ID NO:2; substitution of 139 (R)from SEQ ID NO:1 with 138 (W) from SEQ ID NO:2; substitution of 141-144(FSRF) from SEQ ID NO:1 with 140-143 (QSPH) from SEQ ID NO:2;substitution of 146-149 (EVRE) from SEQ ID NO:1 with 145-148 (AVKD) fromSEQ ID NO:2; substitution of 151 (A) from SEQ ID NO:1 with 150 (V) fromSEQ ID NO:2; substitution of 153 (V) from SEQ ID NO:1 with 152 (I) fromSEQ ID NO:2; substitution of 157-158 (AA) from SEQ ID NO:1 with 156-157(TT) from SEQ ID NO:2; substitution of 160-162 (GDA) from SEQ ID NO:1with 159-161 (ETS) from SEQ ID NO:2; substitution of 164 (A) from SEQ IDNO:1 with 163 (K) from SEQ ID NO:2; substitution of 168 (A) from SEQ IDNO:1 with 167 (E) from SEQ ID NO:2; substitution of 170-171 (YD) fromSEQ ID NO:1 with 169-171 (VE) from SEQ ID NO:2; substitution of 174 (L)from SEQ ID NO:1 with 173 (T) from SEQ ID NO:2; substitution of 177-179(QEK) from SEQ ID NO:1 with 176-178 (KHR) from SEQ ID NO:2; substitutionof 181-182 (GL) from SEQ ID NO:1 with 180-181 (KA) from SEQ ID NO:2;substitution of 184-187 (DVML) from SEQ ID NO:1 with 183-187 (NFIF) fromSEQ ID NO:2; substitution of 199-202 (RPSQ) from SEQ ID NO:1 with198-201 (PKKA) from SEQ ID NO:2; substitution of 204-205 (SS) from SEQID NO:1 with 203-204 (KN) from SEQ ID NO:2; substitution of 209 (W) fromSEQ ID NO:1 with 208 (R) from SEQ ID NO:2; substitution of 211 (S) fromSEQ ID NO:1 with 210 (D) from SEQ ID NO:2; substitution of 213 (T) fromSEQ ID NO:1 with 212 (R) from SEQ ID NO:2; substitution of 215 (Q) fromSEQ ID NO:1 with 214 (V) from SEQ ID NO:2; substitution of 219 (P) fromSEQ ID NO:1 with 218 (G) from SEQ ID NO:2; substitution of 221-222 (SA)from SEQ ID NO:1 with 220-221 (QE) from SEQ ID NO:2; substitution of226-228 (ATP) from SEQ ID NO:1 with 225-228 (VKKS) from SEQ ID NO:2;substitution of 230 (H) from SEQ ID NO:1 with 230(N) from SEQ ID NO:2;substitution of 238-239 (VA) from SEQ ID NO:1 with 238-239 (LR) from SEQID NO:2; substitution of 241-246 (MLLRGA) from SEQ ID NO:1 with 241-246(QEIVSS) from SEQ ID NO:2; substitution of 250-251 (D) from SEQ ID NO:1with 250-251 (K) from SEQ ID NO:2; substitution of 252-254 (ALP) fromSEQ ID NO:1 with 252-254 (NSV) from SEQ ID NO:2; substitution of 256 (N)from SEQ ID NO:1 with 256 (D) from SEQ ID NO:2; substitution of 259-260(AA) from SEQ ID NO:1 with 259-260 (KA) from SEQ ID NO:2; substitutionof 262 (G) from SEQ ID NO:1 with 262 (K) from SEQ ID NO:2; substitutionof 264-267 (SDQ) from SEQ ID NO:1 with 264-267 (TEEE) from SEQ ID NO:2;substitution of 269-271 (QAI) from SEQ ID NO:1 with 269-271 (LDV) fromSEQ ID NO:2; substitution of 275 (Y) from SEQ ID NO:1 with 275 (F) fromSEQ ID NO:2; substitution of 279-280 (VM) from SEQ ID NO:1 with 279-280(FK) from SEQ ID NO:2; and substitution of 282 (K) from SEQ ID NO:1 with282-305 (QSSRAFTNSKKSVTLRKKTKSKRS) from SEQ ID NO:2.

These building block substitutions from D1L3 results in variants of D1,which feature one or more of the following mutations: 1_MS22delinsMSRELAPLLLLLLSIHSALA, L23_A27delinsMRICS, 130_T32delinsVRS,E35_T36delinsES, M38_I47delinsQEDKNAMDVI, Q49_S52delinsKVIK, Y54C,156_Q60delinsIILVM, V62_R63delinsIK, S65_K72delinsSNNRICPI,L74_N76delinsMEK, Q79_T84delinsRNSRRGIT, H86N, V88_V89delinsVI,E91_P92delinsSR, N96_S97delinsNT, R101Q, L103A, V105L,R107_Q110delinsKEKL, A113_S116delinsVKRS, Y118H, D120H,G122_N128delinsYQDGDA, T130S, N132S, A136_I137delinsFV, R139W,F141_F144delinsQSPH, E146_E149delinsAVKD, A151V, V153I,A157_A158delinsTT, G160_A162delinsETS, A164K, A168E, Y170 D171delinsVE,L174T, Q177_K179delinsKHR, G181 L182delinsKA, D184_L187delinsNFIF,R199_Q202delinsPKKA, S204_S205delinsKN, W209R, S211D, T213R, Q215V,P219G, S221_A222delinsQE, A226_P228delinsVKKS, H230N, V238_A239delinsLR,M241_A246delinsQEIVSS, D250K, A252_P254delinsNSV, N256D,A259_A260delinsKA, G262K, 5264_L267delinsTEEE, Q269_I271delinsLDV,Y275F, V279_M280delinsFK, K282delinsQSSRAFTNSKKSVTLRKKTKSKRS.

In some embodiments, a recombinant D1 variant comprises the followingmodifications with respect to SEQ ID NO:1: Y275F, V279_M280delinsFK, andK282delinsQSSRAFTNSKKSVTLRKKTKSKRS (SEQ ID NO: 16). Such variantscomprise a C-terminal tail, and display higher affinity for DNA-lipidcomplexes.

In some embodiments, a recombinant D1 variant comprises the followingamino acid modifications with respect to SEQ ID NO:1:Q79_T84delinsRNSRRGIT (e.g., SEQ ID NO: 10); H86N, and/or,V88_V89delinsVI, and/or E91_P92delinsSR (e.g. SEQ ID NO: 11, whichincludes all three); A136_I137delinsFV (e.g., SEQ ID NO: 12);R199_Q202delinsPKKA, and/or S204_S205delinsKN, and/or W209R, and/orS211D, and/or T213R, and/or Q215V, and/or P219G, and/orS221P_A222delinsQE (e.g., SEQ ID NO: 13, which includes all eight); andA226_P228delinsVKKS (e.g., SEQ ID NO: 14).

In some embodiments, where the D1 variant comprises the substitutionH86N, the variant comprises at least one addition modification, such asa building block substitution or other point mutation described herein(such as the addition of a cationic amino acid or substitution thatresults in resistance to actin).

In some embodiments, the D1 variant comprises at least two adjacent,contiguous, building block substitutions that result in mutations withrespect to SEQ ID NO:1 of: H86N/V88_V89delinsVI (e.g., SEQ ID NO: 11);R199_Q202delinsPKKA/S204_S205delinsKN/W209R/S211D/T213R/Q215V/P219G/S221_A222delinsQE(e.g., SEQ ID NO: 13);Y275FN279_M280delinsFK/K282delinsQSSRAFTNSKKSVTLRKKTKSKRS (SEQ ID NO:16).

In some embodiments, the invention provides a DNase enzyme comprising anamino acid sequence that is at least 80% identical to the amino acidsequence of the enzyme defined by SEQ ID NO:1, and comprising at leastone building block substitution from the amino acid sequence of SEQ IDNO:2, where the DNase enzyme has increased chromatin-degrading activityas compared to the DNase enzyme of SEQ ID NO:1. In some embodiments, theenzyme comprises at least two adjacent building block substitutions fromSEQ ID NO:2. Building block substitutions between D1 (SEQ ID NO:1) andD1L3 (SEQ ID NO:2) are illustrated in FIG. 31.

In some embodiments, the enzyme comprises the following modificationwith respect to SEQ ID NO:1: Q79_T84delinsRNSRRGIT, and optionallycomprises the amino acid sequence of SEQ ID NO: 10.

In some embodiments, the enzyme comprises the following modificationwith respect to SEQ ID NO:1: H86N/V88_V89delinsVI/E91_P92delinsSR, andoptionally comprises the amino acid sequence of SEQ ID NO: 11.

In some embodiments, the enzyme comprises the following modificationswith respect to SEQ ID NO:1: A136_I137delinsFV, and optionally comprisesthe amino acid sequence of SEQ ID NO: 12.

In some embodiments, the enzyme comprises two or more of the followingmodifications with respect to SEQ ID NO:1: R199_Q202delinsPKKA, and/orS204_S205delinsKN, and/or W209R, and/or S211D, and/or T213R, and/orQ215V, and/or P219G, and/or S221_A222delinsQE, and optionally comprisesthe amino acid sequence of SEQ ID NO: 13 (which comprises all eight).

In some embodiments, the enzyme comprises the following modificationswith respect to SEQ ID NO:1: A226_P228delinsVKKS, and optionallycomprises the amino acid sequence of SEQ ID NO: 14.

In some embodiments, the enzyme comprises two or more of the followingmodifications with respect to SEQ ID NO:1: Q79_T84delinsRNSRRGIT, and/orH86N, and/or V88_V89delinsVI, and/or E91_P92delinsSR, and/orA136_I137delinsFV, and/or R199_Q202delinsPKKA, and/or S204_S205delinsKN,and/or W209R, and/or S211D, and/or T213R, and/or Q215V, and/or P219G,and/or S221_A222delinsQE, and/or A226_P228delinsVKKS, and optionallycomprises the amino acid sequence of SEQ ID NO: 15.

In some embodiments, the enzyme comprises two or more the followingmodification with respect to SEQ ID NO:1:Y275F, and/orV279_M280delinsFK, and/or K282delinsQSSRAFTNSKKSVTLRKKTKSKRS, andoptionally comprises the amino acid sequence of SEQ ID NO: 16.

Exemplary D1 variants include those comprising the amino acid sequenceof SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ IDNO:15, and SEQ ID NO:16. The invention in some embodiments, includesderivatives having from 1 to 10 (e.g., 1 to 5) amino acid insertions,deletions, or substitutions with respect to any one of SEQ ID NOS: 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In some embodiments, the D1variant comprises one or more additional block amino acid substitutionsfrom a homologous DNase (e.g., from D1L3). Such block substitutions mayreplace from 2 to 20 amino acids, such as from 3 to 15 amino acids, orfrom 3 to 10 amino acids of the D1 variant, with building block aminoacids from the homologous DNase (e.g., D1L3, SEQ ID NO:2). Buildingblock substitutions between D1 and D1L3 are illustrated in FIG. 31. Insome embodiments, the DNase enzyme comprises an amino acid sequence thatis at least 95% or at least 97% identical to SEQ ID NO:15.

In various embodiments, the enzyme exhibits one or more propertiesselected from: similar or higher affinity for protein-free DNA comparedto the enzyme of SEQ ID NO:1, a higher chromatin degradation activitycompared to the enzyme of SEQ ID NO:1, an enzymatic activity that isresistant to inhibition by actin compared to the enzyme of SEQ ID NO:1,and a combination thereof.

In some embodiments, the D1 variant comprises an N-terminal orC-terminal fusion to a half-life extending moiety, such as albumin,transferrin, an Fc, or elastin-like protein, as described for D1L3. Insome embodiments, the D1 enzyme is dimerized by an immunoglobulin hingeregion.

In various embodiments, the D1 variant exhibits one or more propertiesselected from: the same or higher protein-free DNA (naked DNA)degradation activity, higher chromosomal DNA (chromatin) degradationactivity, similar or improved protease resistance, actin resistance,similar or improved penetration from blood into urine and/or bile, andhigher production levels in vitro expression systems (e.g. Chinesehamster ovary cells and/or Pichia pastoris), and a combination thereof.

In various embodiments, the DNase enzyme comprises an amino acidsequence that is at least 80% identical to the amino acid sequence ofthe enzyme defined by SEQ ID NO:1, and comprises the substitution,insertion, or addition of one or more arginine and/or lysine residues,wherein the DNase enzyme has increased chromatin-degrading activity ascompared to the enzyme of SEQ ID NO:1. In some embodiments, the one ormore arginine and/or lysine residues are amino acid substitutionsselected from substitutions at positions corresponding to positions 31,41, 49, 52, 68, 76, 79, 81, 92, 109, 114, 115, 164, 177, 181, 200, 201,204, 209, 213, 227, 239, 250, 259, 262, and 280 of SEQ ID NO:1.Exemplary modifications at these positions include Q31R, A41K, Q49R,S52K, T68R, N76K, Q79R, D80_A8linsR, A81R, P92R, D109K, V114K, D115R,A164K, Q177K, G181K, P200K, S201K, 5204R, W209R, T213R, A226_T227insK,T227K, A239R, D250K, A259K, G262K, and K280M. In various embodiments,the enzyme comprises substitution and/or insertion of at least two, orat least three, or at least four, or at least five, or at least sixarginine and/or lysine residues. In some embodiments, DNase enzymecomprises an arginine or lysine at one or more positions correspondingto positions 31, 49, 76, 79, 92, 109, 164, 177, 200, 201, 204, 209, 213,and 262 of SEQ ID NO:1, which are optionally selected from Q31R, Q49R,N76K, Q79R, P92R, D109K, A164K, Q177K, P200K, S201K, S204R, W209R,T213R, W209R, and G262K. These positions/substitutions can independentlyprovide better activity, including activity for degrading chromatin.

In various embodiments, if the enzyme comprises a substitution of Q31Ror T227K, the enzyme comprises at least two amino acid modificationsselected from Q31R, A41K, Q49R, S52K, T68R, N76K, Q79R, D80_A81insR,A81R, P92R, D109K, V114K, D115R, A164K, Q177K, G181K, P200K, S201K,S204R, W209R, T213R, A226_T227insK, T227K, A239R, D250K, A259K, G262K,and K280M. In some embodiments, the amino acid substitutions includeQ31R and T227K with respect to SEQ ID NO:1.

In some embodiments, the DNase enzyme has an arginine or lysine atpositions corresponding to positions 31, 49, 79, and 209 of SEQ ID NO:1,and may optionally comprise the following amino acid substitutions:Q31R, Q49K, Q79R, and W209R. These positions harbor an arginine orlysine in murine D1, but not wild-type human D1, and can transferchromatin-degrading activity to human D1.

In some embodiments, the DNase enzyme has an arginine or lysine atpositions corresponding to positions 31, 49, 79, 177, 209, and 262 ofSEQ ID NO:1, and may optionally comprise the amino acid substitutionsQ31R, Q49K, Q79R, A136F, Q177K, W209R, G262K. Four of these positionsharbor an arginine or lysine in rat D1, but not wild-type human D1, andtogether can transfer chromatin degrading activity to human D1.

In some embodiments, the DNase enzyme has a lysine or arginine residueat positions corresponding to positions 31 and 227 of SEQ ID NO:1.

In some embodiments, the DNase enzyme has a lysine or arginine at theposition corresponding to position 164 of SEQ ID NO:1, which isoptionally lysine (A164K).

In various embodiments, the engineered D1 protein contains one or moreamino acid modifications (e.g., substitutions, additions, and deletions)in the actin binding site (e.g. substitution of A136, such as A136F),and thereby hindering or preventing actin binding. As such, theengineered D1 protein can be actin-resistant or substantiallyactin-resistant. See Ulmer et al., PNAS USA Vol. 93, pp 8225-8229(1996). Thus, in various embodiments, the DNase enzyme comprises anamino acid substitution at position 136 with respect to SEQ ID NO:1, andwhich is optionally A136F. Mutations to D1 providing for actinresistance, such as mutations at position 136 (e.g., A136F, orsubstitution of another bulky, hydrophobic, cyclic or aromatic aminoacid at this position), may be combined with any other mutationdescribed herein, including addition of cationic residues or buildingblock substitutions.

In some embodiments, the D1 variant is resistant to actin-inhibition andcapable of degrading chromatin. For example, the D1 variant may comprisethe following amino acid substitutions: Q31R, T227K, and A136F withrespect to SEQ ID NO:1. In some embodiments, the DNase enzyme comprisesthe following amino acid substitutions: A164K and A136F.

In some embodiments, the DNase enzyme comprises a glycosylationconsensus sequence that comprises a N40S substitution and/or an N128Ssubstitution with respect to SEQ ID NO:1. For example, in someembodiments the enzyme comprises the amino acid substitutions: Q31R,N405, A136F, N128S, and T227K with respect to SEQ ID NO:1.

In various embodiments, the D1 variant contains one or more amino acidmodifications (e.g., substitutions, additions, insertions, anddeletions) in the glycosylation sites, and thereby increasing affinitytowards lipid encapsulated DNA. As such, the engineered D1 protein candegrade lipid encapsulated DNA in transfection reactions. See Wilber etal., Mol Ther 6, 35-42 (2002).

In various embodiments, the D1 variant is glycosylated, or may benon-glycosylated particularly where long circulating half-life is notneeded. The amino acid alterations described herein can be made in thecontext of glycosylated or non-glycosylated variants.

In some embodiments, D1 variant is resistant to actin-inhibition andcapable of degrading chromatin within lipid vesciles. Exemplary D1variants comprise one or more amino acid modifications selected from:Q31R, T227K, A136F, N4OD or N40A, and N128D or N128S, with amino acidsnumbered according to SEQ ID NO:1. Such variants may lack glycosylationsites, which are beneficial from a manufacturing persepctive.

In some embodiments, the D1 variant is resistant to actin-inhibition andcapable of degrading chromatin within lipid vesicles, and comprisesamino acid modifications Q31R, T227K, A136F, N40D, and N128D withrespect to SEQ ID NO:1.

Other modifications to D1 can be as described in Pan et al. J Biol Chem,1998, 273, 11701-11708 and in US 2014/0199329, the entire disclosure ofwhich is hereby incorporated by reference in its entirety.

The engineered D1 variant can have an increased or extended serumhalf-life compared to the wild-type D1 protein (SEQ ID NO:1). In someembodiments, the engineered D1 comprises an Fc-fusion domain (e.g. ahinge region, and/or and CH2 domains and CH3 domains of animmunoglobulin). In some embodiments, the engineered D1 is fused toalbumin, e.g., human albumin or a fragment thereof. Albumin can bejoined to at the N-terminus or the C-terminus of the engineered D1enzyme, optionally through an amino acid a linker.

In some aspects, the invention provides pharmaceutical compositionscomprising the recombinant D1L3 and/or D1 variants, or polynucleotidesor vectors encoding the same, and a pharmaceutically acceptable carrier.In various embodiments, the recombinant D1L3 protein variant isformulated for parenteral or pulmonary administration. In someembodiments, the composition is formulated for intravenous,intraarterial, intraperitoneal, intraarticular, intramuscular, topical,or subcutaneous administration, or other route described herein. In someembodiments, the composition comprises both D1L3 and D1, which are eachoptionally engineered variants described herein. In some embodiments,the recombinant D1L3 variant and the recombinant D1 variant areformulated for parenteral or pulmonary administration.

The term “pharmaceutically acceptable carrier” includes, but is notlimited to, any carrier that does not interfere with the effectivenessof the biological activity of the ingredients and that is not toxic tothe patient to whom it is administered. Examples of suitablepharmaceutical carriers are well known in the art and include phosphatebuffered saline solutions, water, emulsions, such as oil/wateremulsions, various types of wetting agents, sterile solutions etc. Suchcarriers can be formulated by conventional methods and can beadministered to the subject at a suitable dose. Preferably, thecompositions are sterile. These compositions may also contain adjuvantssuch as preservative, emulsifying agents and dispersing agents.Prevention of the action of microorganisms may be ensured by theinclusion of various antibacterial and antifungal agents.

Routes of administration include, for example: intradermal,intramuscular, intraperitoneal, intraarticular, intravenous,subcutaneous, intraarterial, oral, sublingual, pulmonary, ortransdermal. In some embodiments, the administering is effected orallyor by parenteral injection or infusion. In some embodiments, the routeof administration is topical, including as eye drops or mouth wash.

In still other aspects, the invention provides a method of making apharmaceutical composition for reducing or preventing neutrophilextracellular trap (NET) accumulation. In these embodiments, theinvention employs a genetically modified mouse deficient in D1 and D1L3activity, and heterologous expression of a G-CSF polynucleotide (e.g.,in hepatocyte cells) or induction of a sustained endogenous G-CSFexpression (e.g., via repetitive administration of microbial compounds).This mouse model accumulates NETs and rapidly develops NET-relatedvascular occlusions. In these embodiments, the invention comprisesadministering a candidate NET inhibitor or candidate DNase enzyme(including a D1L3 or D1 variant in accordance with this disclosure) tothe genetically-modified mouse, and selecting a NET inhibitor or DNaseenzyme that reduces the accumulation of NETs. The selected inhibitor orenzyme is formulated (as described) for administration to a humanpatient.

One skilled in the art recognizes standard methods for generating doubleknockout Dnase1^(−/−)Dnase1l3^(−/−) mice. Detailed descriptions can befound in, for example, European Application No. EP 17152528.0.

In some aspects, the invention provides a method for treating a subjectin need of neutrophil extracellular trap (NET) degradation. The methodcomprises administering a therapeutically effective amount of D1L3and/or D1 (which may include variants described herein, or in someembodiments one or more wild type enzymes, including for D1L3 and D1combination therapy) according to this disclosure. The D1L3 or D1 may beadministered as pharmaceutical compositions comprising the recombinantprotein, or in some embodiments comprising the encoding DNA or RNA orvectors comprising the same.

In some embodiments, the method comprises administering atherapeutically effective amount of a D1L3 or variant thereof and a D1or variant thereof. The D1L3 or variant thereof, and the D1 or variantthereof, may be administered in a single pharmaceutical composition orseparate pharmaceutical compositions. In some embodiments, the ratio ofD1L3 or variant thereof to D1 or variant thereof is in the range of100:1 to 1:100 by mass, or 10:1 to 1:10 by mass, and is optionally about1:1.

In some embodiments, the subject exhibits impaired NET degradationand/or exhibits pathological NET accumulation. In some embodiments, thesubject has a chronic or acute inflammatory disorder. In someembodiments, the subject has an acute or chronic infection.

In various embodiments, the present invention pertains to the treatmentof diseases or conditions characterized by the presence or accumulationof NETs. Such diseases or conditions include, but are not limited to,diseases associated with chronic neutrophilia (e.g., an increase in thenumber of neutrophils), neutrophil aggregation and leukostasis,thrombosis and vascular occlusion (e.g. sickle cell disease),ischemia-reperfusion injury (e.g. midgut volvulus, testicular torsion,limb ischemia reperfusion, vital organ ischemia-reperfusion, organtransplantation), surgical and traumatic tissue injury, an acute orchronic inflammatory reaction or disease, an autoimmune disease (e.g.systemic lupus erythematosus (SLE), lupus nephritis, rheumatoidarthritis, vasculitis, systemic sclerosis), cardiovascular disease(e.g., myocardial infarction, stroke, atherosclerosis, venousthromboembolism, including thrombolytic therapy), metabolic disease(e.g., diabetes), systemic inflammation (e.g., systemic inflammatoryresponse syndrome (SIRS), sepsis, septic shock, disseminatedintravascular coagulation (DIC), and thrombotic microangiopathy (TMA)),inflammatory diseases of the respiratory tract (e.g. cystic fibrosis,chronic obstructive pulmonary disease (COPD), acute lung injury (ALI),smoke induced lung injury, transfusion induced lung injury (TRALI),acute respiratory distress syndrome (ARDS), and asthma, atelectasis,bronchitis, empyema), renal inflammatory diseases (acute and chronickidney diseases, including acute kidney injury (AKI) and chronic kidneydisease (CKD), inflammatory diseases related to transplated tissue (e.g.graft-versus-host disease) and cancer (e.g. leukemia, tumor metastasis,and solid tumors).

In some embodiments, the subject has or is at risk of NETs occludingductural systems. The present invention can be administered to a subjectto treat pancreatitis, cholangitis, conjunctivitis, mastitis, dry eyedisease, obstructions of vas deferens, or renal diseases. In suchembodiments, the method comprises administering a therapeuticallyeffective amount of a D1L3 or variant thereof, and/or a therapeuticallyeffective amount of D1 or a variant thereof. For example, the subjectmay be administered an enzyme comprising an amino acid sequence having80% or more sequence identity with the enzyme defined by SEQ ID NO:2,and an enzyme comprising an amino acid sequence having 80% or moresequence identity with the enzyme defined by SEQ ID NO:1. In someembodiments, the enzymes comprise the amino acid sequence of the enzymedefined by SEQ ID NO:2 and the amino acid sequence of the enzyme definedby SEQ ID NO:1.

In some embodiments, the ductal system is bile duct, tear duct,lactiferous duct, cystic duct, hepatic duct, ejaculatory duct, parotidduct, submandicular duct, major sublingual duct, bartholin's duct,cerebral aqueduct, pancreas, mammary gland, vas deferens, ureter,urinary bladder, gallbladder, and liver. For example, the subject mayhave pancreatitis, cholangitis (e.g., primary sclerosing cholangitis),conjunctivitis, mastitis, dry eye disease, an obstruction of the vasdeferens, or renal disease. In some embodiments, the DNase enzyme isadministered by intravenous, intraarterial, or intraperitonealadministration. In various embodiments, the DNase when applied, forexample, intravenously, will be present in enzymatically active form invarious ductal systems, such as in bile fluid.

In some embodiments, the subject has or is at risk of NETs accumulatingon endothelial surfaces (e.g. surgical adhesions), the skin (e.g.wounds/scarring), or in synovial joints (e.g. gout and arthritis). Thepresent invention can be administered to a subject to treat a conditioncharacterized by an accumulation of NETs on an endothelial surface suchas, but not limited to, a surgical adhesion. In various embodiments, thepresent invention can be administered to a subject to treat a conditioncharacterized by an accumulation of NETs on skin such as, but notlimited to, wounds and scars. In certain embodiments, the presentinvention can be administered to a subject to treat a conditioncharacterized by an accumulation of NETs in a synovial joint such as,but not limited to, gout and arthritis.

In some embodiments, the subject has or is at risk of a vascularocclusion comprising NETs. In such embodiments, the method comprisesadministering a therapeutically effective amount of a D1L3 or variantthereof, and/or a therapeutically effective amount of D1 or a variantthereof. For example, the subject may be administered an enzymecomprising an amino acid sequence having 80% or more sequence identitywith the enzyme defined by SEQ ID NO:2. In some embodiments, the enzymecomprises the amino acid sequence of the enzyme defined by SEQ ID NO:2.In some embodiments, the subject is further administered an enzymecomprising an amino acid sequence having 80% or more sequence identitywith the enzyme defined by SEQ ID NO:1, such as the enzyme defined bySEQ ID NO:1.

In some embodiments, the subject has a condition relating to NETs asdescribed in WO 2016/118476 and U.S. Pat. No. 9,642,822, which arehereby incorporated by reference in its entirety.

In various embodiments, the subject has a disease that is or has beentreated with wild-type DNases, including D1 and streptodornase. Suchdiseases or conditions include thrombosis, stroke, sepsis, lung injury,atherosclerosis, viral infection, sickle cell disease, myocardialinfarction, ear infection, wound healing, liver injury, endocarditis,liver infection, pancreatitis, primary graft dysfunction, limb ischemiareperfusion, kidney injury, blood clotting, alum-induced inflammation,hepatorenal injury, pleural exudations, hemotorax, intrabiliary bloodclots, post pneumatic anemia, ulcers, otolaryngological conditions, oralinfections, minor injuries, sinusitis, post-operative rhinoplasties,infertility, bladder catheter, wound cleaning, skin reaction test,pneumococcal meningitis, gout, leg ulcers, cystic fibrosis, Kartegener'ssyndrome, asthma, lobar atelectasis, chronic bronchitis, bronchiectasis,lupus, primary cilliary dyskinesia, bronchiolitis, empyema, pleuralinfections, cancer, dry eyes disease, lower respiratory tractinfections, chronic hematomas, Alzheimer's disease, and obstructivepulmonary disease.

In certain embodiments, the present invention pertains to the treatmentof diseases or conditions characterized by deficiency of D1, deficiencyof D1L3, and deficiency of D1 and D1L3. In some cases, the subject has amutation in the Dnase1 and/or the Dnase1l3 gene. Such subjects can alsohave an autoimmune disease (e.g., SLE, systemic sclerosis) or aninflammatory disease. In some cases, the subject has an acquiredinhibitor of D1(e.g., anti-DNase1-antibody and actin) and/or the D1L3(e.g., anti-DNase1l3-antibody). Such subjects can also have anautoimmune disease (e.g., SLE, systemic sclerosis) or an inflammatorydisease (e.g., sepsis and ischemia-reperfusion injury).

In some embodiments, the subject has cystic fibrosis, and the DNasecomposition is administered by pulmonary delivery.

In various embodiments, for example, where the subject is at risk of, orexhibits symptoms of, NET-related vascular occlusion, the protein orcomposition (e.g., comprising D1L3, D1, or a variant of D1L3 or D1,including variants described herein) may be administered intravenously,intramuscularly, subcutaneously, or intraarterially.

In various embodiments, the subject is monitored for the NET-degradingactivity of blood, plasma, or serum. For example, the subject may bemonitored for about one week to about four weeks, to reduce the risk ofvascular or ductal occlusion by NETs, or NET-related damage to organs.In some embodiments, the subject may receive from one to fouradministrations over the course of from one week to one month (or 1-2weeks), as needed to prevent or reduce intravascular NET accumulation(including NET-related intravascular occlusions) during an acuteinfection or inflammatory event. In other embodiments, the subject mayreceive continuous infusion over the course of from one hour to onemonth (or 1-2 weeks). In some cases, the subject may receive at leastone, e.g., 1, 2, 3, 4 or more infusions daily, for example (withoutlimitation), in the case of critical illness such as sepsis, stroke, ormyocardial infarction.

The treatment of disease or conditions relating to NETs may involveadministration of a D1 variant, a D1L3 variant, or a combinationcomprising a D1 variant and a D1L3 variant. In some embodiments, two ormore of variants of the D1 protein described herein are administered. Insome embodiments, two or more of variants of the D1L3 protein describedherein are administered.

The present D1 protein variants and D1L3 protein variants can beadministered as a prophylactic therapy, such as before the onset of oneor more symptoms of the disease or condition.

As used herein, “treatment” or “treating” or “treated” refers totherapeutic treatment wherein the object is to slow (lessen) anundesired physiological condition, disorder or disease, or to obtainbeneficial or desired clinical results. Beneficial or desired clinicalresults include, but are not limited to, alleviation of symptoms;diminishment of the extent of the condition, disorder or disease;stabilization (i.e., not worsening) of the state of the condition,disorder or disease; delay in onset or slowing of the progression of thecondition, disorder or disease; amelioration of the condition, disorderor disease state; and remission (whether partial or total), whetherdetectable or undetectable, or enhancement or improvement of thecondition, disorder or disease. Treatment includes eliciting aclinically significant response without excessive levels of sideeffects. In other embodiments, “treatment” or “treating” or “treated”refers to prophylactic measures, wherein the object is to delay onset ofor reduce severity of an undesired physiological condition, disorder ordisease, such as, for example is a person who is predisposed to adisease (e.g., an individual who carries a genetic marker for a diseasesuch as lupus).

In the methods of the invention, therapy is used to provide a positivetherapeutic response with respect to a disease or condition. By“positive therapeutic response” is intended an improvement in thedisease or condition, and/or an improvement in the symptoms associatedwith the disease or condition. For example, a positive therapeuticresponse would refer to one or more of the following improvements in thedisease: (1) a reduction in neutrophil extracellular traps; (2) areduction in intravascular clots; (3) a reduction in ductural clots; (4)a reduction of accumulation of NETs in synovial joints; (5) an increasein the degradation of neutrophil extracellular traps; (6) an increase indegradation of extracellular protein-free DNA; (7) an increase indegradation of extracellular chromosomal DNA or protein-bound DNA; (8)an increase in the degradation of neutrophil extracellular traps in thepresence of autoantibodies; (9) a reduction in tissue/organinflammation; (10) a reduction in tissue/organ injury; (11) a reductionin tissue/organ atrophy; (12) an increased patient survival rate; and(13) some relief from one or more symptoms associated with the diseaseor condition.

Positive therapeutic responses in any given disease or condition can bedetermined by standardized response criteria specific to that disease orcondition. Clinical response can be assessed for changes in vascularocclusion and the presence of NETs using screening techniques such asmagnetic resonance imaging (MRI) scan, ultrasound scan, histology, andcounting of NETs in the circulation. In addition to these positivetherapeutic responses, the subject undergoing therapy may experience thebeneficial effect of an improvement in the symptoms associated with thedisease.

Dosage regimens are adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. Parenteral compositions may beformulated in dosage unit form for ease of administration and uniformityof dosage. Dosage unit form as used herein refers to physically discreteunits suited as unitary dosages for the subjects to be treated; eachunit contains a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier.

In various embodiments, the present invention provides an expressionvector, comprising a nucleic acid encoding a wild type or D1L3 or D1variant described herein, which can find use for genetic therapy.Genetic therapy can be useful for chronic illness such as a geneticdeficiency of D1L3 and/or D1 (such as a deleterious mutation). Invarious embodiments, the expression vector comprises DNA or RNA. Invarious embodiments, the expression vector is a mammalian expressionvector.

In some embodiments, expression vectors comprise a nucleic acid encodingthe protein variants operably linked to an expression control region, orcomplement thereof, that is functional in a host cell (e.g.,prokaryotic, eukaryotic, or mammalian cell). The expression controlregion is capable of driving expression of the operably linked blockingand/or stimulating agent encoding nucleic acid such that the blockingand/or stimulating agent is produced in a human cell transformed withthe expression vector.

Expression control regions are regulatory polynucleotides (sometimesreferred to herein as elements), such as promoters and enhancers, thatinfluence expression of an operably linked nucleic acid. An expressioncontrol region of an expression vector of the invention is capable ofexpressing operably linked encoding nucleic acid in a human cell. In anembodiment, the expression control region confers regulatable expressionto an operably linked nucleic acid. A signal (sometimes referred to as astimulus) can increase or decrease expression of a nucleic acid operablylinked to such an expression control region. Such expression controlregions that increase expression in response to a signal are oftenreferred to as inducible. Such expression control regions that decreaseexpression in response to a signal are often referred to as repressible.Typically, the amount of increase or decrease conferred by such elementsis proportional to the amount of signal present; the greater the amountof signal, the greater the increase or decrease in expression.

Expression systems functional in human cells are well known in the art,and include viral systems. Generally, a promoter functional in a humancell is any DNA sequence capable of binding mammalian RNA polymerase andinitiating the downstream (3′) transcription of a coding sequence intomRNA. A promoter will have a transcription initiating region, which isusually placed proximal to the 5′ end of the coding sequence, andtypically a TATA box located 25-30 base pairs upstream of thetranscription initiation site. The TATA box is thought to direct RNApolymerase II to begin RNA synthesis at the correct site. A promoterwill also typically contain an upstream promoter element (enhancerelement), typically located within 100 to 200 base pairs upstream of theTATA box. An upstream promoter element determines the rate at whichtranscription is initiated and can act in either orientation. Ofparticular use as promoters are the promoters from mammalian viralgenes, since the viral genes are often highly expressed and have a broadhost range. Examples include the SV40 early promoter, mouse mammarytumor virus LTR promoter, adenovirus major late promoter, herpes simplexvirus promoter, and the CMV promoter.

Where appropriate, gene delivery agents such as, e.g., integrationsequences can also be employed. Numerous integration sequences are knownin the art (see, e.g., Nunes-Duby et al., Nucleic Acids Res. 26:391-406,1998; Sadwoski, J. Bacteriol., 165:341-357, 1986; Bestor, Cell,122(3):322-325, 2005; Plasterk et al., TIG 15:326-332, 1999; Kootstra etal., Ann. Rev. Pharm. Toxicol., 43:413-439, 2003). These includerecombinases and transposases. Examples include Cre (Sternberg andHamilton, J. Mol. Biol., 150:467-486, 1981), lambda (Nash, Nature, 247,543-545, 1974), FIp (Broach, et al., Cell, 29:227-234, 1982), R(Matsuzaki, et al., J. Bacteriology, 172:610-618, 1990), cpC31 (see,e.g., Groth et al., J. Mol. Biol. 335:667-678, 2004), sleeping beauty,transposases of the mariner family (Plasterk et al., supra), andcomponents for integrating viruses such as AAV, retroviruses, andantiviruses having components that provide for virus integration such asthe LTR sequences of retroviruses or lentivirus and the ITR sequences ofAAV (Kootstra et al., Ann. Rev. Pharm. Toxicol., 43:413-439, 2003). Inaddition, direct and targeted genetic integration strategies may be usedto insert nucleic acid sequences encoding the chimeric fusion proteinsincluding CRISPR/CAS9, zinc finger, TALEN, and meganuclease gene-editingtechnologies.

All cited references are herein expressly incorporated by reference intheir entirety.

EXAMPLES Example 1 Host DNases Prevent Vascular Occlusion by NeutrophilExtracellular Traps

Platelet and fibrin clots occlude blood vessels in hemostasis andthrombosis. Here we report a non-canonical mechanism for vascularocclusion based on neutrophil extracellular traps (NETs), antimicrobialDNA-fibers released by activated neutrophils. We show that two DNases,DNase1 and DNase1-like 3, degrade NETs in circulation during episodes ofmurine inflammation. In the absence of both DNases, intravascular NETsform clots that obstruct blood vessels and cause organ damage. Vascularocclusions in patients with severe bacterial infections are associatedwith a defect to degrade NETs ex vivo and the formation of intravascularNET-clots. DNase1 and DNase1-like 3 are independently expressed and thusprovide dual host protection against deleterious effects of NETs ininflammation.

Inflammation is an essential host response to control invading microbesand heal damaged tissues (1). Uncontrolled and persistent inflammationcauses tissue injury in a plethora of inflammatory disorders.Neutrophils are the predominant leukocytes in acute inflammation. Duringinfections neutrophils generate extracellular traps (NETs), lattices ofDNA-filaments decorated with toxic histones and enzymes that immobilizeand neutralize bacteria (2). The relevance of NETs in host defense isillustrated by the fact that extracellular DNases serve as virulencefactors in several pathogenic bacteria (3, 4).

However, inappropriately released NETs may harm host cells due to theircytotoxic, proinflammatory, and prothrombotic activity (5-7). Indeed,NETs are frequently associated with inflammatory or ischemic organdamage and the therapeutic infusion of DNases limits host injury invarious animal models (8, 9).

How the host degrades NETs in vivo to limit tissue damage duringepisodes of inflammation is poorly understood. Earlier work has shownthat DNase1 in serum digests the DNA-backbone of NETs in vitro (10). Weanalyzed serum from wild type mice by zymography and detected twoenzymatically active DNases, DNase1 and DNase1-like 3 [(DNase1l3) (FIG.1A). Both enzymes are members of the DNase1 protein family, but differin their origin and substrate affinity. DNase1 is expressed bynon-hematopoietic tissues and preferentially cleaves protein-free DNA(11,12). DNase1l3, also known as DNase γ, is secreted by immune cellsand targets DNA-protein-complexes, such as nucleosomes (11,13). Wegenerated mice that lacked DNA-degrading activity in serum due to acombined deficiency of DNase1 and DNase1l3 (FIG. 1A). In vitro generatedNETs remained intact after exposure to DNase1/DNase1l3−/− sera, whereassera from wild type, DNase1−/−, and DNase1l3−/− mice degraded NETs (FIG.1B and FIG. 1C). We used a hepatocyte-specific expression plasmid inconjunction with hydrodynamic gene delivery to stably express the cDNAof DNase1 or DNase1l3 in the liver of DNase1/DNase1l3−/− mice. Giventhat both enzymes contain a secretory protein signal sequence (11), thisapproach restored the activity of DNase1 or DNase1l3 in circulation(FIG. 1D) and the capacity of sera from DNase1/DNase1l3−/− mice todegrade NETs (FIG. 1E and FIG. 1F). Taken together, these data show thattwo independently expressed host enzymes, DNase1 and DNase1l3, degradeNETs in vitro.

To test the requirement of DNase1 and DNase1l3 for NET-degradation invivo, we aimed to chronically stimulate wild type, DNase1−/−,DNase1l3−/−, and DNase1/DNase1l3−/− mice with the granulocyte-colonystimulating factor (G-CSF). G-CSF triggers neutrophilia, a hallmark ofacute inflammation, and stimulates a subpopulation of neutrophils tospontaneously release NETs ex vivo (14). We cloned the cDNA of CSF3,which encodes G-CSF, into the hepatocyte-specific expression plasmid.Hydrodynamic injection of wild type mice with the CSF3-plasmid resultedin chronically elevated levels of G-CSF in plasma (FIG. 5A).Consequently, the neutrophil blood count steadily increased andspontaneously formed NETs were detected in blood smears (FIG. 2A andFIG. 2B). We furthermore observed an increased number of residentneutrophils in vital organs and splenomegaly (FIG. 5B and FIG. 5C).Importantly, CSF3-injected wild type mice grew normally, did not developorgan injury, and did not show macroscopic signs of distress or abnormalbehavior (FIG. 5D and FIG. 5E). Collectively, these data suggest thatchronic neutrophilia with concomitant NET formation is well tolerated inwild type mice.

Next, we stably expressed CSF3 in the liver of DNase1−/−, DNase1l3−/−,and DNase1/DNase1l3−/− mice. Mice with a single deficiency in DNase1 orDNase1l3 did not show signs of distress (not shown), whereas all micewith a combined deficiency died within 6 days after CSF3-injection (FIG.2C). DNase1/DNase1l3−/− mice receiving the control plasmid lacking CSF3survived without showing any abnormalities (FIG. 2C, not shown). Weco-expressed DNase1 or DNase1l3 with CSF3 in DNase1/DNase1l3−/− mice toinduce neutrophilia and NETs and simultaneously restore DNase1 orDNase1l3 in circulation. Expression of either DNase was sufficient forDNase1/DNase1l3−/− mice to survive without showing any signs of distress(FIG. 2D). DNase1/DNase1l3−/− mice co-expressing CSF3 with a controlplasmid lacking DNase1 and DNase1l3 died within 5 days after genedelivery (FIG. 2D). The mortality in these mice was preceded by arapidly progressing hypothermia, which was evidenced as a strongdecrease in peripheral body temperature within 8 hours before exitus(FIG. 2E). Hypothermia was accompanied with hemolytic anemia shown byreddish plasma and urine and reduced blood hemoglobin (FIG. 2F and FIG.2G). Abundant schistocytes in blood smears indicated that the hemolyticanemia was caused by erythrocyte fragmentation (FIG. 2H). Furthermore,we detected elevated plasma levels of lactate dehydrogenase (LDH), livertransaminases, and the renal injury markers blood urea nitrogen andcreatinine, which indicated multiple organ damage (FIG. 21, FIG. 6A, andFIG. 6B). Coexpression of DNase1 or DNase1l3 with CSF3 maintained thebody temperature and integrity of erythrocytes and organs (FIG. 2E toFIG. 2I; FIG. 6A and FIG. 6B). In conclusion, these data indicate thateither DNase1 or DNase1l3 is required to prevent host injury duringchronic neutrophilia.

The histological analysis of DNase1/DNase1l3−/− mice with chronicneutrophilia showed intravascular hematoxylin-positive clots withentrapped erythrocytes that fully or partially occluded blood vessels inlungs, liver, and kidneys (FIG. 3A and B; FIG. 6C and FIG. 6D).Expression of DNase1 or DNase1l3 in circulation prevented these vascularocclusions. The hematoxylin-positive clots showed an abundantlight-violet staining pattern that was sporadically speckled with adark-violet staining of individual leukocyte nuclei, suggesting thatdecondensed DNA is a main clot component (FIG. 3A). Given that nuclearbreakdown and unfolding of tightly packed chromatin is a hallmark ofNET-formation (15), we stained the hematoxylin-positive clots forNET-markers. We observed a robust staining with fluorescentdouble-stranded DNA-intercalating dyes and antibodies against chromatin(FIG. 7A). The co-localization of decondensed chromatin with theneutrophil granule-derived enzyme myeloperoxidase, antimicrobialcathelicidin peptides, and the NET-surrogate marker citrullinatedhistones confirmed that the clots were composed of NETs (FIG. 3C, FIG.7B and FIG. 7C). To identify components of canonical thrombi, we stainedNET-clots for fibrin and von Willebrand factor (vWF), a protein storedin the secretory vesicles of platelets and the vascular endothelium.NET-clots were very heterogeneous in their vWF and fibrin content (FIG.3D and FIG. 3E). Cross-sections of NET-clots were covered on averagewith 46% of vWF, while 9% of NET-clots did not stain for vWF (FIG. 3Eand FIG. 3F). Fibrin was largely absent and detected in less than 23% ofthe occluded vessels (FIG. 3E). These data are in line with findingsthat NETs serve as a fibrin-independent scaffold to immobilize plateletsand erythrocytes in vitro (6). The absence of vWF and fibrin in someclots suggests that NETs may be sufficient for vascular occlusion. Tocorroborate this notion, we aimed to generate NET-clots from pureneutrophils in vitro. We isolated neutrophils from blood and inducedNET-formation, while exposing the cells to shear forces to mimic bloodflow. In this setup, we observed macroscopically visible andDNase-sensitive clots (FIG. 8A), which resembled the appearance ofNET-clots within the murine vasculature (FIG. 8B). Now, we depletedplatelets from the circulation and pharmacologically inhibited thrombinto block fibrin formation in CSF3-expressing DNase1/DNase1l3−/− mice.Unlike DNase1- or DNase1l3-expression, neither anti-thrombotic treatmentcould prevent mortality in these animals (FIG. 3H). Collectively, thedata suggest that clots of NETs are sufficient to obstruct blood vesselsduring chronic neutrophilia in DNase1/DNase1l3−/− mice.

The formation of NET-clots in DNase1/DNase1l3−/− mice is associated withfeatures of infection-induced thrombotic microangiopathies (TMAs) anddisseminated intravascular coagulation in patients, includingschistocytes, hemolytic anemia, and organ failure due to vascularocclusions. We analyzed plasma from TMA patients with hemolytic-uremicsyndrome due to an infection with Shiga toxin-producing Escherichia coli[STEC-HUS, (16)]. Sepsis and septic shock was a frequent complication inthese patients (17). NETs generated in vitro remained intact afterexposure to patient plasma collected in the acute disease state, whereasplasma from patients in remission degraded NETs (FIG. 9A and FIG. 9B).The data indicate an acquired and temporary defect in NET-degradation,thus extending previous reports (18,19). Of note, the STEC-HUS patientswere effectively treated with a regimen that included infusion of plasmafrom healthy donors (17), a source of DNases, which potentially restoredNET-degradation.

Large aggregates of NETs are reportedly formed in the synovial fluid andpancreatic ducts of patients (21,22), but have not yet been described inother tissues. Therefore, we aimed to identify intravascular aggregatesof NETs in patients with severe inflammatory diseases. We screened lungtissue collected at autopsy from patients with acute respiratorydistress syndrome and/or sepsis (FIG. 10). We detectedhematoxylin-positive clots in blood vessels of two septic patients (FIG.11A and FIG. 11C). In both cases, clots were comprised of chromatin andMPO (FIG. 11B and FIG. 11D), indicating that NETs can form intravascularclots in human sepsis.

Septicemia is a potent and rapid trigger of intravascular NET-formationin mice (7). We therefore hypothesized that a defect in NET-degradationmay aggravate the disease. Indeed, we observed that mice with a combineddeficiency of DNase1 and DNase1l3, but not wild type mice, were highlysusceptible to low doses lipopolysaccharide and heat-killed E. coli(FIG. 4A). Similar to neutrophilic DNase1/DNase1l3−/− mice, bloodanalysis of septic DNase1/DNase1l3−/− mice showed hemolytic anemia andhematuria (FIG. 4B and FIG. 4C), along with increased levels of plasmaLDH and schistocytes in blood smears (FIG. 4D and FIG. 4E). Furthermore,we detected abundant partially or fully occluded blood vessels in thelung (FIG. 4F and FIG. 4G). Detailed analysis of partially occludedvessels revealed clots of NETs within the vascular lumen (FIG. 4H). Infully occluded vessels the NET-clots were congested with entrappederythrocytes and leukocytes (FIG. 41; FIG. 12A and FIG. 12B).Importantly, hepatic expression of DNase1 or DNase1l3 inDNase1/DNase1l3−/− mice prevented vascular occlusion and restored thewild type phenotype. Taken together, these data indicate thatcirculating DNase1 or DNase1l3 prevent the formation of NET-clots andhost injury in septicemia.

In summary, while platelets and fibrin form hemostatic clots andpathological thrombi (23), our data introduces NET-clots as anon-canonical mechanism for vascular occlusion in inflammatory states.Similar to fibrin strands, NETs are ultra-large and stable molecules(6). At high concentrations, such as found in chronic neutrophilia orsepticemia, intravascular NETs may form clots, which are sufficient insize to obstruct blood vessels and thus cause damage to erythrocytes andorgans. To maintain blood and tissue integrity during inflammation, thehost independently expresses DNase1 and DNase1l3 as a dual protectionsystem against intravascular NETs. However, acquired and genetic defectsin these host factors may delay the degradation of NETs and thusprecipitate disease. Acquired defects may involve DNase1-inhibition bymonomeric actin externalized from damaged tissue and inactivation ofDNase1l3 by serum proteases (11). Mutations in DNase1 and DNase1l3 havebeen identified in patients and are associated with systemic lupuserythematosus (SLE), a systemic autoimmune disease (24,25).Interestingly, DNase1 and DNase1l3-deficient mice spontaneously developSLE-like disease with age (13,26). NETs are composed of prominentautoantigens and neutrophils from SLE patients have an increasedcapacity to release NETs (27). A reduced clearance capacity may increasethe half-life NETs and thus promote the autoimmune disease (10,27). Inconclusion, defects in host DNases may bring forward the deleteriouseffects of NETs in a plethora of inflammatory diseases.

Consequently, DNase1- and DNase1l3-mediated NET-degradation providesattractive therapeutic opportunity for patients suffering frominflammatory conditions. To test whether DNase1 and DNase1l3 degradeNETs by the same or via distinct mechanisms, we cross-linked proteins inNETs using fixative. Fixed NETs were efficiently degraded by DNase1l3−/−sera, but were resistant to degradation by DNase1-−/− sera (FIG. 13A andFIG. 13B). In line with these results, exposure of NETs to recombinantDNase1 or DNase1l3 showed that both DNases degrade naive NETs (FIG. 13Cand FIG. 13D), whereas fixed NETs are resistant to DNase1l3 activity(FIG. 13E and FIG. 13F). Collectively, these in vitro data suggest thatDNase1 and DNase1l3 degrade NETs via distinct mechanisms.

Finally, we compared the activity of DNase1 and DNase1l3 in thecirculation of mice to humans. To discriminate DNase1 and DNase1l3activity, we generated antibodies against human DNase1 (α-hDNase1),which block DNase1 activity in plasma from normal healthy donors (NHD)(FIG. 14A). Heparin is a known inhibitor of DNase1l3 and was used toblock the DNA-degrading activity by DNase1l3 (FIG. 14B). Murine plasmashowed approximately 10-fold higher total DNase activity (FIG. 14C),DNase1 activity (FIG. 14D), and DNase1l3 activity (FIG. 14E) than humanplasma. Furthermore, DNase1l3 in murine plasma, but not in human plasma,degrades NETs efficiently (FIG. 14F and FIG. 14G). The data suggest thatthe concentration of DNase 1l3 in human circulation is not sufficient todegrade NETs. Therefore, enabling DNase1l3-mediated NET-degradation inpatients a novel and promosing therapeutic strategy for inflammatorydiseases.

Materials and Methods

Patient Plasma

Citrated plasma samples were obtained from patients during the STEC-HUSoutbreak 2011 in Germany (16). The criteria for inclusion in our studywere positive identification of STEC/enterohemorrhagic E. coli or bloodydiarrhea, platelet count 150×109/L or decreased by 25% in 1 week,evidence of hemolysis (LDH above the normal limit, haptoglobin below thenormal limit, or the presence of schistocytes) and acute kidney injurystage

Human tissue

Human tissues were fixed at room temperature overnight in 5%phosphate-buffered formalin. All tissues were collected at autopsy andoriginally submitted for diagnostic purposes to us and studied inaccordance with national ethical principles. The cause of death of thepatients is indicated in FIG. 10.

Mice

All mice were on a C57BL/6 genetic background. We crossed previouslydescribed DNase1−/− and DNase1-like 3−/− mice (26,28) to generateDNase1/DNase1l3-−/− mice. This strain of DNase1−/− mice has beenrecently reported to contain an off-target mutation in the DNase1overlapping gene Trap1/Hsp75, which encodes a mitochondrial chaperone(29). We therefore included an alternative DNase1−/− strain (30) togenerate DNase1/DNase1l3−/− mice for control experiments (FIG. 15A-FIG.15C)

Detection of DNase1 by Denaturing Polyacrylamide Gel ElectrophoresisZymography (DPZ)

We performed DPZ, as previously described with modifications (19). Inbrief, sodium dodecyl sulfate (SDS)-polyacrylamide gels were preparedwith 4% (v/v) stacking gels without DNA and 10% (v/v) resolving gelscontaining 200 μg/ml of salmon testes DNA (Sigma- Aldrich, Germany). Forthe detection of DNase1, 0.5 μl of murine serum were mixed with 14.5 μlof water and 5 μl SDS gel-loading buffer (BioRad, Germany). The mixturewas boiled for 5 minutes and loaded onto the gels. Electrophoresis wascarried out at 120 V using Tris/glycine electrophoresis buffer (25 mMTris, 192 mM glycine, 0.1% (w/v) SDS, pH 8.7). After electrophoresis,proteins were refolded by incubating the gels overnight at 37° C. in arefolding buffer containing 5% (w/v) milk powder, 10 mM Tris/HCl pH 7.8,3 mM CaCl₂, 3 mM MgCl₂, 100 U/mL penicillin, and 100 μg/mL streptomycin.The gels were transferred to a refolding buffer without milk powder andincubated for additional 24 hours at 37° C. DNA was labeledfluorescently with 1× SYBR Safe (Thermo Scientific, Germany) andfluorescent images of gels were recorded using a fluorescence scanner(Molecular Imager FX, BioRad, Germany).

Detection of DNase1l3 by DPZ

For the detection of DNase1l3, 2 μl of serum were mixed with 12 μl ofwater, 5 μl SDS gelloading buffer, and 1 μl of beta-mercaptoethanol(BME, Sigma-Aldrich). BME reduces the disulfide bridges of DNase1, whichcauses its inactivation (11). The mixture was boiled for 5 minutes andloaded onto the gels. Electrophoresis was carried out as described forDNase1. SDS was removed by washing the gels with 10 mM Tris/HCl pH 7.8for 30 minutes at 50° C. twice. The proteins were refolded by incubatingthe gels for 48 hours at 37° C. in a refolding buffer containing 10 mMTris/HCl pH 7.8, 1 mM BME, 100 U/mL penicillin and 100 μg/mLstreptomycin. The gels were then incubated for additional 48 hours at37° C. in refolding buffer supplemented with 3 mM CaCl2 and 3 mM MnCl2.The addition of Mn²⁺ is required to enable efficient degradation ofprotein-free DNA by DNase1l3 (11). DNA was labeled and imaged asdescribed for DNase1.

Detection of Total DNase Activity by Single Radial Enzyme Diffusion(SRED) Assay

To measure total DNase activity, we dissolved 55 μg/ml DNA from salmontestes in a buffer containing Mn²⁺ (20 mM Tris-HCl pH 7.8, 10 mM MnCl₂,2 mM CaCl₂, and 2× SYBR Safe). The DNA solution was heated at 50° C. for10 minutes and mixed with an equal volume of 2% ultra-pure agarose(Thermo Scientific). The mixture was poured into plastic trays andstored at room temperature until solidification. Two μl of murine serumwere loaded into wells of 1.0 mm diameter. Gels were incubated for 4hours at 37° C. in a humid chamber. The DNA fluorescence of the gels wasrecorded with a fluorescence scanner.

In Vitro NET-Degradation Assay

NET-degradation was analyzed as previously described (19). Purifiedhuman neutrophils in serum-free DMEM were seeded to sterile 96-wellplates (Falcon, BD Technologies, Germany) coated with 0.001% polylysine(Sigma) at a concentration of 5×10⁴ cells per well. To induceNET-formation, neutrophils were activated with 100 nM phorbol12-myristate 13- acetate (PMA; Sigma-Aldrich) for 4 h at 37° C. with 5%CO₂ and humidity. We added phosphate buffered saline (PBS) and storedthe 96-well plates overnight at 4° C. NETs were washed with PBS, treatedfor 5 minutes with PBS containing 0.5% Triton X-100, and washed with PBSagain. NETs were incubated with 10% murine serum or 10% citrated humanplasma supplemented with 10 μM PPACK (Santa Cruz, Heidelberg, Germany)in HBSS with divalent cations (HBSS+; Thermo Scientific). NETs wereallowed to be degraded for 3 hours at 37° C. with 5% CO2 and humidity.We discarded supernatants and stopped NET-degradation by adding 2% PFAin PBS for 1 hour at room temperature. PFA was discarded andnon-degraded NETs were labeled fluorescently by adding 2 μM of thefluorescent DNA dye SytoxGreen (Thermo Scientific) in PBS. Images offluorescently stained NETs were acquired with an inverted fluorescencemicroscope (Axiovert 200M, Zeiss, Germany). We quantified nondegradedNETs by recording the fluorescence intensity with a plate reader(Excitation: 485 nm; Emission: 535 nm) or by measuring the area coverageof NETs in microscopy pictures using ImageJ software (NIH, USA).

Preparation of In Vivo Expression Vectors

We used the pLIVE plasmid (Mirus Bio, USA) to express proteins in mice.The vector enables a long-lasting and hepatocyte-specific expression ofproteins. We generated pLIVE plasmids with murine DNase1, DNase1l3, andCSF3. For murine DNase1, a PCR of the cDNA (Genbank Accession NumberNM010061) was performed using the pair of primers DNase1-F 5′-GTCGACATGCGGTACACAGG (SEQ ID NO:27) and DNase1-R 5′-CTCGAGTCAGATTTTTCTGAGTGTCA (SEQ ID NO:28) containing SalI and XhoIrestriction sites. For DNase1l3, a PCR of the cDNA (Genbank AccessionNumber AF047355) was performed using the pair of primers DNase1l3-F5′-GAAGTCCCAGGAATTCAAAGATGT (SEQ ID NO:29) and DNase1l3-R5′-GCGTGATACCCGGGAGCGATTG (SEQ ID NO:30) containing BamHI and Sadrestriction sites. Both cDNAs were cloned using the T4 ligase (NewEngland Biolabs, Germany) into the multi-cloning site (MCS) of the pLIVEvector, which was predigested with the appropriate enzymes. The pLIVEvector containing DNase1l3 was subjected to site-directed mutagenesiswith the pair of primers mutDNase1l3-F 5′-AGTCGACTCCCGGCCACCATGTCCCTGCA(SEQ ID NO:31) and its complementary mutDNase1 1l3-R 5′-TGCAGGGACATGGTGGCCGGGAGTCGACT (SEQ ID NO:32) in order to match theconsensus Kozak sequence and optimize protein expression. The generationof the pLIVE vector containing the cDNA of CSF3 was outsourced (EurofinsGenomics, Germany). The cDNA of CSF3 (Genbank Accession Number BC120761)was inserted in the MCS between restriction sites SalI and XhoI.Sequence of all the generated vectors was confirmed by double strandedDNA sequencing. As a control we used the parental pLIVE plasmid withoutadditional inserts. All plasmids were purified using PureLink HiPurePlasmid Maxiprep Kit and potential contaminations of endotoxin wereremoved using High Capacity Endotoxin Removal Spin Columns (both ThermoScientific).

In Vitro Expression of Murine DNase1 and DNase1l3

Plasmids containing the cDNA for DNase1 or DNase1l3, described elsewherewere transfected into Human Embryonic Kidney (HEK) cells withLipofectamine 3000 (Thermo Scientific) in serum-free conditions withDMEM media (Thermo Scientific) supplemented with 10% KnockOut serumreplacement (Thermo Scientific). After 72 hours, supernatants werecollected, centrifuged at 500×g for 10 minutes, sterile filtered, andconcentrated by ultracentrifugation with 3K columns (Amicon Ultra,Millipore, Darmstadt, Germany).

In Vivo Gene Expression

The pLIVE-plasmids containing DNase1, DNase1l3, CSF3, or empty controlplasmids were administered to mice via hydrodynamic tail vein injection.In brief, 50 μg of plasmid were diluted in 0.9% saline in a volumeequivalent to 10% of the body mass of the mouse. Mice were anaesthetizedwith isoflurane and the plasmid solution was then injected intravenouslyover 5 to 8 seconds via the tail vein. In rare cases, mice did not fullyrecover from the injection within the first 24 hours and these animalswere excluded from the study. For coexpression studies, 50 μg of theCSF3-plasmid were mixed with 50 μg of the empty control plasmid, or theplasmids containing DNase1 and DNase1l3. The solution containing bothplasmids was administered via hydrodynamic tail vein injection.

Chronic Neutrophilia

Female mice at 4 weeks or 8-12 weeks of age were injected with 50 μg ofthe pLIVE-plasmid containing CSF3 to induce G-CSF overexpression andneutrophilia. Mice were monitored every 8 hours during the first weekafter injection, and daily afterwards. Temperature was measured in theperianal area by a contactless infrared-thermometer (Etekcity, Germany).For survival studies, mice were euthanized and scored as “non-surviving”if the animals showed signs of distress (no spontaneous movement, closedeyes, occasional gasping). In all cases these signs of distress wereaccompanied with a rapidly progressing and severe hypothermia, definedas decrease in body temperature of ≥4° C. compared to the bodytemperature before the plasmid injection, and with hematuria.Non-hypothermic mice did not show any signs of distress and wereeuthanized at the end of the experiment. For organ, blood, and urinecollection, we analyzed four groups of three D1/D1L3−/− mice. Mice ineach group were injected with a mixture of CSF3 with empty plasma,DNase1, or DNase1l3. Each group of mice was euthanized for biosamplecollection, when the first animal of the group showed signs of distress,severe hypothermia, and hematuria. This time point was defined as“exitus” and occurred within 3 to 6 days after the injection.

Platelet Depletion

Mice received an intraperitoneal injection of 2 μg/g platelet-depletingantibody or isotype control antibody (both Emfret, Germany). Thistreatment depletes >95% of platelets from circulation. The treatment wasstarted 24 hours after the injection of CSF3 and repeated every 48 hoursuntil completion of the experiment.

Thrombin Iinhibition

Powdered dabigatran etexilate (Pradaxa, Boehringer Ingelheim, Germany)was mixed with normal chow powder (Altromin, Germany) at a dose of 40mg/g. Pellets were prepared by mixing the powder with distilled waterand allowed to dry at room temperature. The feeding of dabigtran wasstarted 24 hours after the injection of CSF3. WT mice fed with thedabigratan diet for 1 day showed a 6.48±1.19-fold (Mean±SD, N=4)increased activated partial thromboplastin time when compared to micereceiving normal chow without dabigratan (Student t-test; P<0.001).

Preparation of Bacteria for Septicemia

Escherichia coli (XEN 14, Perkin Elmer) was grown overnight in lysogenybroth media containing 50 μg/ml kanamycin. Bacteria were pelleted bycentrifuging at 4000×g for 10 minutes, washed with and resuspended inPBS. Aliquots of 1.5×10⁹ bacteria/ml were incubated at 70° C. for 15minutes to heat-kill the bacteria. Aliquots stored at −20° C. untilfurther use.

Septicemia

Male mice at 8-15 weeks of age received three daily intraperitonealinjections of 1 μg/g of LPS from Salmonella enterica serotypethyphimurium (Sigma-Aldrich) in 0.9% saline. Along with the third LPSinjection, mice received an intravenous injection of 1.5×107 heat-killedE. coli. The shown survival time indicates the time after the injectionof E. coli. Blood and organs were collected at the time of euthanasia.Insufficient biosamples were obtained from two animals (1×D1/D1L3−/−+Ctrl, 1× D1/D1L3−/−+D1L3) to perform the complete analysisshown in FIG. 4A-FIG. 4I. Mice were euthanized and scored as“non-surviving” if the animals showed signs of severe distress(irresponsiveness to touch). All non-surviving mice showed hematuria andpaleness of extremities. All surviving mice were euthanized and scoredas “surviving” 24 hours after the intravenous injection of heat-killedE. coli.

Murine Blood, Plasma, and Tissue Collection

Blood was collected by submandibular puncture or by the retroorbitalsinus. Blood was collected into 200 μl serum tubes (Monovette; Sarstedt,Germany) and 200 μl EDTA tubes (GK 150, KABE labortechnik, Germany).Plasma was obtained by collecting the supernatant of blood aftercentrifugation at 3000×g for 15 minutes. Serum was obtained by allowingthe blood to clot for 1 hour at room temperature followed by acentrifugation for 15 minutes at 3000×g. Serum and plasma samples werestored in aliquots at −20° C. until further use. For organ analysis,mice were perfused by intracardiac infusion of PBS. Organs werecollected and fixed for 24 hours in 4% PFA at 4° C. Fixed organs wereembedded in paraffin.

Analysis of Blood and Plasma

LDH, AST, ALT, creatinine and BUN in plasma were quantified by usingstandardized kits (Biotron Diagnostics, CA, USA) following themanufacturer instructions. Mouse G-CSF was quantified with a QuantikineELISA Kit (R&D, United Kingdom). Hemoglobin in EDTA blood was quantifiedby an automated hemocytometer (Idexx ProCyte Dx Hematology Analyzer,Netherlands). To quantify neutrophils by FACS, whole blood was incubatedon ice for 15 minutes with 0.2 μg of phycoerythrine-labelled anti-mouseCD11b (M1/70, Biolegend, Germany) and 0.5 μig of Alexa Fluor 488anti-mouse Ly6G (1AB, Biolegend, Germany). The blood was then dilutedwith 0.5 ml PBS and analyzed with a FACSCalibur (BD Bioscience, USA).Blood smears of EDTA-anticoagulated blood were prepared onpolylysine-coated slides (Hecht Assistant, Germany). After air-dryingwere incubated for 1 minute in methanol supplemented with 1 μMSytoxGreen on dry ice or stained with Giemsa using commercial kit (InVitro Diagnostikum, Germany).

Immunofluorescence Stainings

Paraffin-embedded sections were de-waxed, rehydrated, and subjected toantigen retrieval for 25 minutes at 100° C. in citrate buffer (10 mMsodium citrate, 0.1% Tween, pH 6). Thereafter, sections were blocked for30 minutes with 2.5% normal goat serum (Vector, United Kingdom) followedby incubation with a mouse-on-mouse blocking kit (Vector) for one hour.The sections were then incubated over night at 4° C. with 2 μ/ml of theprimary antibody against MPO (A0398, Agilent, Germany), CRAMP(PA-CRLP-100, Innovagen, Sweden), citrullinated histone 3 (ab5103,Abcam, United Kingdom), fibrin [clone 59D8], or the complex of histoneH2A, H2B, and DNA to detect chromatin (2). Sections were incubated withanti-rabbit and anti-mouse IgG antibodies conjugated with AlexaFluor488or AlexaFluor555 (all Thermo Scientific) for 1 hour. After washing, DNAwas labeled with 1 μg/ml DAPI for 2 minutes. Autofluorescence wasquenched by a 25-minute incubation with Sudan Black (0.1% in 70%ethanol), and sections were mounted with Fluoromount G (SouthernBiotech, USA). Images of fluorescently labeled sections were acquiredwith an inverted fluorescence microscope (Axiovert 200M, Zeiss, Germany)or a confocal microscope (TCS SPS, Leica, Germany). Images of NET-clotswere quantified using software (ImageJ). A vWF or fibrin staining areaof less than 3% of NET-clots was considered negative.

Immunohistochemistry

After antigen retrieval, deparaffinized sections were blocked for 30minutes with 2.5% normal horse serum (Vector). The sections wereincubated over night at 4° C. with 2 μg/ml of the primary antibodyagainst neutrophil elastase (ab68672, Abcam). After washing, sectionswere stained using the anti-rabbit IgG-AP kit, (ImmPRESS, Vector)according to manufacturer's instruction. Sections were counterstainedwith hemalum (Merck, Germany) and mounted with Neo-Mount media (Merck).Images of stained sections were acquired with an inverted microscope(Axiovert 200M, Zeiss).

Generation of NET-Clots In Vitro

Purified human neutrophils were seeded to tissue culture plates at 3×10⁷cells/ml in DMEM supplemented with 2% BSA. The cells were activated with0.1 μM PMA for 4 hours at 37° C., 5% CO₂ with humidity and rotatingconditions (300 rpm). We used unstimulated neutrophils and neutrophilsactivated with PMA in the presence 10 U/ml recombinant human DNase1(dornase alpha, Roche, Germany) as controls. NET-clots were fixed with2% PFA overnight at 4° C. Fixed NET-clots were embedded in paraffin andsections were stained for NETs as described above.

Statistical Evaluation

Data are shown as mean±standard deviation. Statistical analysis wasperformed using Prism Software (GraphPad, USA) and included Student'st-test, one-way and two-way ANOVA followed by Bonferroni's multiplecomparison test, and Log-rank test. Results were considered significantat P<0.05.

Movies

Chronic neutrophilia is tolerated in wild type mice. A movie generatedshows the wild type mice one week after the injection of aCSF3-expression plasmid (CSF3) or a control plasmid lacking CSF3 (Ctrl).Both animals show normal behavior and no signs of distress.

Chronic neutrophilia causes distress in mice lacking DNase1 andDNase1l3. The movie shows DNase1/DNase1l3−/− mice co-expressing CSF3with a control plasmid (CSF3/Ctrl), with DNase1 (CSF3/D1,), or withDNase1l3 (CSF3/D1L3). Mice expressing circulatory DNases, DNase1 orDNase1l3, show normal behavior. Mice lacking circulatory DNases(CSF3/Ctrl) show signs of distress (no spontaneous movement, closedeyes, occasional gasping).

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Example 2 Development and use of DNase1L3 and Variants for TherapyIntroduction

Uncontrolled inflammation causes a plethora of conditions (1).Neutrophil extracellular traps (NETs) are DNA filaments decorated withtoxic proteins (2). NETs protect against microbial infection, butexcessive NET formation triggers and exacerbates inflammatory andautoimmune diseases (3). DNase1 (D1), a DNA-degrading enzyme found inblood, has been widely used to disarm NETs in vitro and in vivo. Indeed,infusion of recombinant human D1, an FDA-approved drug (dornase alpha)for cystic fibrosis (CF), prevents the pathological effects of NETs invarious disease models (3), supporting the use of D1 for prophylactictherapy.

DNase1-like 3 is Novel Drug Candidate for NET-Associated Diseases

In Example 1, we found that in addition to D1, DNase1-like 3 (D1L3) alsodegrades NETs. Both enzymes maintain blood and tissue homeostasis duringinflammatory responses. In brief, we showed that NETs are generatedwithin blood vessels during neutrophilia, a hallmark of inflammation,under sterile as well as infectious conditions. D1 and D1L3 cleave NETsand thus prevent the aggregation of DNA-filaments of NETs to vascularclots. In the absence of D1 and D1L3, clots ofNETs obstruct blood flowand cause organ damage and death. NET-clots are formed independently ofplatelets or fibrin and are therefore resistant to conventionalanti-thrombotic therapy. Thus, we uncovered clots of NETs as a novelmechanism for vascular occlusion and D1L3 a new prophylactic therapyagainst NETs (see Example 1).

D1 and D1L3 belong, along with DNase1-like 1 (D1L1) and DNase1-like 2(D1L2), to the DNase1-protein family (4). D1 and D1L3 are expressed in avariety of species including, humans, primates, and rodents. D1 ispredominantly expressed in the gastrointestinal tract and exocrineglands (5), whereas hematopoietic cells, namely macrophages anddendritic cells produce D1L3 (6, 7). We found both DNases at lowconcentrations in serum of mice and humans (Example 1, FIG. 14), but theorigin of D1 and D1L3 in circulation is unknown (7, 8). D1preferentially cleaves protein-free DNA (e.g. bacterial DNA, plasmidDNA), whereas D1L3 targets chromatin, the complex of DNA and histones,which is commonly found in the nucleus of eukaryotic cells (7, 9, 10).D1 activity is inhibited upon binding to monomeric actin and sensitiveto physiological salt concentrations (11-13). In addition, D1 isglycosylated at N40 (corresponds to N18 in the mature enzyme withoutsignal peptide) and N128 (N106), which makes the enzyme resistant toinactivation by serum proteases (9). In contrast, D1L3 lacksglycosylation and actin-binding sites, which causes its susceptibilitytowards several proteases and resistance towards actin, respectively(9).

D1 was first discovered in body fluids of the gastrointestinal tract andurine, taking part in the digestion of DNA from food intake. We observedin the course of this disclosure that systemically administered DNase1(e.g. through intraperitoneal or intravenous injection) has a relativelyshort half-life in circulation and is secreted in an enzymaticallyactive form into the urine and, unexpectedly into bile. Its secretioninto the bile fluid opens up the opportuntity to target extracellularDNA and/or NETs in the biliary duct via systemic DNase1 therapy.

D1L3 was first described in the context of programmed cell death (14).D1L3 features two nuclear localization sites (NLS1, NLS2). NLS2 isembedded within a C-terminal tail that is unique to D1L3 and not presentin D1. Both NLS target the enzyme to the nucleus during apoptosis (14).Indeed, intracellular D1L3 is required for fragmentation of nuclear DNAwithin apoptotic and necrotic cells in vivo (15). D1L3 requires itsC-terminal tail to degrade extracellular DNA, namely lipid-encapsulatedDNA, such as found in transfections, and chromatin within apoptoticbodies (7, 10). In transfections, cDNA and cationic lipids form acomplex that penetrates through the plasma membrane of target cells.D1L3 interferes with transfections and the C-terminal tail is criticalfor this function (10). Apoptotic bodies, lipid vesicles filled withchromatin from apoptotic cells, are physiological substrates of D1L3(7). Here, the C-terminal tail enables D1L3 to penetrate through lipidmembranes of apoptotic bodies and degrade the chromatin load.Importantly, the C-terminal tail is also required for the degradation oflipid-free extracellular chromatin by D1L3 (7). The C-terminal tail isbelieved to be critical for the functions of D1L3 in the extracellularspace.

While hD1 is effectively targeting NETs in animal models, mainly mice,it has shown limited or no therapeutic effect in clinical trials of CF(16) and emphysema (17), respectively. NETs are formed in CF (18),indicating that wild-type hD1 is not optimal for targeting NETs inpatients. Hyperactive variants have been generated to circumvent thetherapeutic limitations of D1, but their clinical manufacturing ischallenging (12). Notably, NET-DNA contains histones and is structurallyorganized like chromatin (2). Thus, we speculated that the efficientdegradation of chromatin is required for an optimal therapy againstNETs.

A combinational therapy of hD1 and tissue plasminogen activator (tPA,alteplase) of emphysema patients has been described (17). The data arein line with our hypothesis because tPA enables D1 to degrade chromatinin vitro (19). U.S. Pat. No. 9,642,822, which is hereby incorporated byreference, further discloses combination therapy with tPA and D1 fortherapeutic targeting of NETs (20). However, treatment with tPA iscommonly associated with an increased risk of bleeding due to itsfibrinolytic activity (17). D1L3 may provide a safer strategy fortargeting NETs without putting patients at risk for bleeding, but atherapy with D1L3 protein has not been explored.

Engineering of DNase1 and DNase1-like 3 Variants through Amino AcidTransfer

In this Example we generated variants of D1 and D1L3 that are engineeredfor therapy against extracellular chromatin (including NETs). To designvariants, we hypothesized that the capacity of human D1L3 to degradechromatin is encoded by individual amino acids and/or amino acidsequences, which are absent in human D1. Furthermore, we speculated thattransferring these individual amino acids and/or an amino acid sequencefrom human D1L3 to D1 generates hyperactive D1 variants with increasedchromatin-degrading activity. Similarly, enzymatic properties (e.g.glycosylation) can be transferred from D1 to D1L3. To test ourhypothesis, we employed conventional amino acid mutations and developeda novel building-block (BB) technology to engineer variants of DNasesfrom the DNase1-protein family.

To produce variants of D1 and D1L3, we used in vitro expression systems[e.g. Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK)cells, Pichia pastoris]. Enzymatic activity was characterized using thedegradation of high-molecular weight (HMW)-DNA as a readout (FIG. 16).We chose two types of HMW-DNA: (a) double-stranded DNA (dsDNA) that wasisolated from salmon testes. The degradation of HMW-dsDNA was analyzedby zymography; (b) chromatin of purified nuclei from HEK293 cells. Toanalyse its degradation, HMW-chromatin was first incubated with theDNase variants, followed by DNA isolation and visualization via agarosegel electrophoresis (AGE). Chromatin degradation was detected by lowmolecular weight (LMW)-DNA. In this setup, wild-type human D1 (SEQ IDNO: 1) has approximately 100-fold higher activity to degrade dsDNA thanwild-type D1L3 (SEQ ID NO: 2), whereas wild-type D1L3 degrades chromatinwith approximately 100-fold more efficiently than wild-type D1 (FIG.16). We found synergistic effects of D1 and D1L3 in degrading chromatin,but not dsDNA. If D1 and D1L3 are mixed at a ratio of 10:1 or 1:1, weobserved increased chromatin degradation, compared to each D1 or D1L3alone (FIG. 16). Thus, we screened for D1 and/or D1L3 variants, whichshow increased chromatin and/or dsDNA degrading activity.

Selected D1 and D1L3 variants were further tested to degradeintravascular NETs, using an in vivo-expression system for members ofthe D1-protein family. See PCT/EP2018/051444, which is herebyincorporated by reference in its entirety. The system is based on twocomponents, (a) an expression plasmid that enables the stable in vivotransfection of hepatocytes with cDNA of D1, D1L3, and variants thereofin recipient mice, and (b) Dnase1^(−/−)Dnase1l3^(−/−) deficient mice,which are characterized by the absence of DNase activity in serum. Inbrief, the cDNA of candidate DNases is cloned into the hepaticexpression vector. Next, the vector is co-expressed inDnase1^(−/−)Dnase1l3^(−/−)deficient mice along with the cDNA of murineCsf3, which encodes for the granulocyte colony-stimulating factor(G-CSF). The expression of Csf3 induces neutrophilia with concomitantintravascular NET-formation, which in turn causes the aggregation ofundigested NETs within blood vessels and death in DNase1^(−/−)Dnase1l3⁻⁻mice. To identify drug candidates, we screened for D1, D1L3, andvariants thereof that efficiently degrade NETs and thus preventmorbidity and mortality in this model.

Results

Engineering DNase1 Variants through Conventional Amino AcidSubstitutions

Our central hypothesis is that the capacity of human D1L3 to degradechromatin is mediated by amino acids that are not present in D1.Sequence alignment showed that 44% of the amino acids in human D1 andhuman D1L3 are identical (FIG. 17). We exclusively mutated only variableamino acids (56% non-shared amino acids) to generate D1 and DL3variants.

D1L3 is a cationic protein with an isoelectric point of 9.35, whereas D1and the other members are more basic proteins. We therefore speculatedthat the cationic nature of D1L3 enables the enzyme to degradechromatin. Through sequence alignment, we identified 37 arginine orlysine residues in the variable amino acids, which are absent in D1(FIG. 17). These amino acid residues are located at the amino acidpositions 29 (position 9 with the signal peptide), 39 (9), 47 (7), 50(30), 66 (46), 74 (54), 77 (57), 80 (60), 81 (61), 92 (72), 109 (89),114 (94), 115 (95), 163 (143), 176 (156), 180 (160), 199 (179), 200(180), 203 (183), 208 (188), 212 (192), 226 (206), 227 (207), 239 (219),250 (230), 259 (239), 262 (242), 280 (260), 285 (265), 291 (271), 292(272), 297 (277), 298 (278), 299 (279), 301 (281), 303 (283), 304 (284)with respect to SEQ ID NO: 2.

Twenty-six of these additional arginine or lysine residues in D1L3correspond to the following amino acid positions in D1 (SEQ ID NO: 1):31 (position 9 with the signal peptide), 41 (19), 49 (40), 52 (30), 68(46), 76 (54), 79 (57), 81 (59), 92 (70), 109 (87), 114 (92), 115 (93),164 (142), 177 (155), 181 (159), 200 (178), 201 (179), 204 (182), 209(187), 213 (191), 227 (205), 239 (217), 250 (228), 259 (237), 262 (240),and 280 (260). In addition, D1L3 features 3 sites of insertions ofarginine or lysine residues: R80, K226 and a C-terminal tail (afteramino acid position 282 in SEQ ID NO: 2), which features 3 arginine and6 lysine residues.

We aimed to transfer the arginine and lysine residues that are part ofthe core body of D1L3 to D1 by introducing the following mutations intowild-type D1 (FIG. 18): Q31R, A41K, Q49R, S52K, T68R, N76K, Q79R,D80_A8linsR, A81R, P92R, D109K, V114K, D115R, A164K, Q177K, G181K,P200K, S201K, S204R, W209R, T213R, A226_T227insK, T227K, A239R, D250K,A259K, G262K, and K280M with respect to SEQ ID NO:1, thus generating 28variants of D1.

In this Example, we were able to generate D1 variants that contain 23 ofthe 28 amino acid substitutions. We expressed the cDNA of theseD1-variants in HEK293 cells and analyzed the dsDNA and chromatindegrading activity in the culture supernatants. We observed that thefollowing amino acid modifications partially increased the chromatindegrading activity, while no major differences in dsDNA degradation wereobserved (FIG. 19): Q31R, Q49K, N76K, Q79R, P92R, D109K, A164K, Q177K,P200K, S201K, S204R, T213R. Interestingly, mouse and rat D1 (SEQ ID NO:4), which share 79% and 78% of the amino acids of human D1,respectively, feature several of the analyzed amino acid substitutions,including Q31R, Q49K, and Q79K (FIG. 20). Analysis of supernatants fromHEK293 cells expressing murine D1 and human D1 showed that murine D1 hasan increased activity to degrade chromatin, when compared to human D1(FIG. 21). We injected Dnase1^(−/−)Dnase1l3^(−/−) mice with a mixture ofCsf3 and murine D1 (mDnase1, SEQ ID NO: 3), human D1 (hDnase1, SEQ IDNO: 1), or an empty control plasmid (Ctrl). Mice receiving the controlplasmid died within 7 days, whereas the expression of murine D1 provideda full protection for at least 28 days (FIG. 21). Unexpectedly, theexpression of human D1 showed only a partial protection from death andmore than half of the animals died within 14 days (FIG. 21), indicatingthat human D1 is less efficient in degrading intravascular NETs thanmurine D1. We now mutated Q31R, Q49K, Q79R, and W209R in human D1 togenerate a murine-like D1-variant (SEQ ID NO: 5). A total of sixmutations (Q31R, Q49K, Q79R, Q177K, W209R, G262K) were made to generatea rat-like D1-variant (SEQ ID NO: 6). Both rodent-like D1 variants werecloned into the hepatocyte expression vector and delivered along withCsf3 into Dnase1^(−/−)Dnase1l3^(−/−) mice. We observed that gene therapywith the murine- and rat-like variant of D1 provided a full protectionagainst NET-mediated vascular occlusions and death (FIG. 22).Importantly, in vitro expression of the rodent-like human D1-variantsshowed an increased activity to degrade chromatin (FIG. 22).Collectively, the data illustrate that (a) human D1 has a limitedcapacity to degrade NETs in vivo; (b) transferring multiple cationicamino acids from human D1L3 to human D1 generates hyperactive variants.

Several attempts have been made to generate hyperactive variants of D1(6, 12, 13, 21-29). We found 184 mutations, which altered 82 of the 282amino acids of wild-type human D1 (FIG. 23). Alignment of amino acidsequences of wild-type human D1 and human D1L3 revealed threegain-of-function mutations in human D1 that are already present inwild-type human D1L3 (FIG. 23): Q31R (corresponds to Q9R in D1 withoutsignal peptide), T227K (T205K), and A136F (A114F). While the mutationsQ31R and T227K may alter the DNA-binding properties of hD1 and thuscause hyperactivity, A136F confers resistance to inhibition by monomericactin (21-24, 26, 29).

We designed a new D1-variant that carries the combination ofQ31R/T227K/A136F (SEQ ID NO: 7) and compared its enzymatic activity towild-type D1 (SEQ ID NO: 1), as well as to D1-variants carrying singlemutations in Q31R, T227K, A136F, or a combined mutation in Q31R andT227K (SEQ ID NO: 8). We characterized the dsDNA and chromatin-degradingactivity in the culture supernatants of transfected HEK293 cells. Weobserved no major differences in dsDNA-degrading activity withD1-variants carrying single or combinational mutations of Q31R and T227K(FIG. 24). Interestingly, chromatin was partially and fully degraded inA136F mutants and the combinational mutation of Q31R/T227K/A136F,respectively (FIG. 24). Next, we tested whether the Q31R/T227K/A136Fvariant of D1 is capable of preventing NET-mediated lethal vascularocclusions in neutrophilic Dnase1^(−/−)Dnase1l3^(−/−). All micereceiving a control plasmid without DNases died, whereas all miceexpressing the D1-variant (SEQ ID NO: 7) survived until the end of theexperiment without showing signs of distress (FIG. 24). Using purifiedproteins, we observed that the triple amino acid substitution increasesthe chromatin degrading activity approximately 10-20 fold compared towild-type D1 without detectable differences in dsDNA degrading activity(FIG. 25). Chromatin from nuclei and NETs contains actin (30, 31). Thedata suggest that chromatin degradation by D1 variants requires cationicamino acid substitutions as well as mutations that confer actinresistance—two features which are present in wild-type D1L3. To furthercorroborate this notion, we generated a D1 variant which features thecombination of the mutations A164K and A136F (SEQ ID NO: 9). Indeed, weobserved a strong increase in chromatin degrading activity in the D1variant with both mutations (FIG. 26), supporting our hypothesis.

Development of Building Block Engineering of DNase1 -Protein FamilyMembers

D1L3 features three sites that contain additional amino acids: theC-terminal tail starting after Q282 (NH₂-SSRAFTNSKKSVTLRKKTKSKRS —COOH)(SEQ ID NO:33), and at two sites within the enzyme at S79/R80 and atK226 (FIG. 17). The 23 amino acids of the C-terminal tail of D1L3 havebeen attached to the C-terminus of D1 (10, 28). We observed that theinsertion of an arginine-residue at position 226 of DNase1(A226_T227insK) generated a D1-variant with reduced enzymatic activityto degrade dsDNA, while no such effect was observed with thesubstitution T227K (FIG. 27). Thus, an insertion of a K/R-residue goesalong with a risk of reducing D1 function. The insertion of a chargedamino acid may influence the local protein structure. Given that D1 is aglobular enzyme that comprises one amino acid chain [FIG. 28, (27)], lit is conceivable that such local alteration may render the whole enzymeinactive. Indeed, numerous non-conservative mutations throughout the D1amino acid sequence inactivate the enzyme (12, 25, 26). We hypothesizedthat the transfer of local protein structures by implanting not onlysingle arginine and lysine residues but also the neighboring amino acidssequences reduces the risk of inactivation. We searched within D1 andD1L3 for conserved amino acids in the vicinity of A226_T227insK that canbe used as anchors for the insertion. We identified a D223/T224/T225motif and a conserved T229 in D1 as N-terminal and C-terminal anchors,respectively (FIG. 27). We replaced 3 amino acids within D1 (ATP) with 4amino acids, including K226, from D1L3 (VKKS) in silico. Expression ofthe cDNA of the new D1-variant (A226_P228delinsVKKS) in HEK239 cellsrevealed a functionally active enzyme with a similar dsDNA-degradingactivity, when compared to wild-type D1 (FIG. 27). The data suggest thatthe variable amino acids between conserved amino acids areinterchangeable between D1 and D1L3.

We conceptualized a building block-technology to transfer enzymaticproperties from one member of the D1-protein family to another. Thefollowing cardinal steps characterize the technology (FIG. 29):

-   -   (1) Provide protein-protein alignment of donor and recipient        DNase    -   (2) Identify variable amino acid or amino acid sequence for        transfer (building block)    -   (3) Identify conserved amino acids in donor and recipient DNase        that are located up and downstream of building block (anchors),        respectively.    -   (4) Replace cDNA encoding for building block between C- and        N-anchors in recipient DNase, with cDNA between the anchors in        donor DNase.    -   (5) Synthesize cDNA of chimeric DNase, followed by in vitro/in        vivo expression into a recipient organism that is preferably        deficient in both donor and recipient DNase (e.g. CHO cells or        Dnase1^(−/−)Dnase1l3^(−/−) mice).        Engineering DNase1 Variants through Building Block Technology

A multiple-species alignment of D1 and D1L3 from human, mouse, rat, andchimpanzee (FIG. 30), showed that N- and C-terminal anchors areconserved among these species. These anchor amino acids or amino acidsequences flank 62 building blocks of variable amino acids and aminoacid sequences, which include the amino acid sequence in D1 (ATP) andD1L3 (VKKS) from the above-mentioned experiments as building blocks #49(FIG. 31).

The transfer of these building blocks from D1L3 into D1 generatesD1-variants with the following mutations (FIG. 31):1M_S22delinsMSRELAPLLLLLLSIHSALA, L23_A27delinsMRICS, 130_T32delinsVRS,E35_T36delinsES, M38_I47delinsQEDKNAMDVI, Q49_S52delinsKVIK, Y54C,156_Q60delinsIILVM, V62_R63delinsIK, S65_K72delinsSNNRICPI,L74_N76delinsMEK, Q79_T84delinsRNSRRGIT, H86N, V88_V89delinsVI,E91_P92delinsSR, N96_S97delinsNT, R101Q, L103A, V105L,R107_Q110delinsKEKL, A113_S116delinsVKRS, Y118H, D120H,G122_N128delinsYQDGDA, T130S, N132S, A136_I137delinsFV, R139W,F141_F144delinsQSPH, E146_E149delinsAVKD, A151V, V153I,A157_A158delinsTT, G160 A162delinsETS, A164K, A168E, Y170_D171 delinsVE,L1 74T, Q177_K179delinsKHR, G181_L182delinsKA, D184_L187delinsNFIF,R199_Q202delinsPKKA, S204_S205delinsKN, W209R, S211D, T213R, Q215V,P219G, S221_A222delinsQE, A226_P228delinsVKKS, H230N, V238_A239delinsLR,M241_A246delinsQEIVSS, D250K, A252_P254delinsNSV, N256D,A259_A260delinsKA, G262K, S264_L267delinsTEEE, Q269_I271delinsLDV,Y275F, V279_M280delinsFK, and K282delinsQSSRAFTNSKKSVTLRKKTKSKRS. Thefollowing D1L3-variants are generated if the building blocks aretransferred from D1 to D1L3 (FIG. 31):M1_A20delinsMRGMKLLGALLALAALLQGAVS, M21_S25delinsLKIAA,V28_S30delinsIQT, E33_S34delinsET, Q36_I45delinsMSNATLVSYI,K47_K50delinsQILS, C52Y, 154_M58delinsIALVQ, 160_K6ldelinsVR,S63_I70delinsSHLTAVGK, M72_K74delinsLDN, R77_T84delinsQDAPDT, N86H,V88_I89delinsVV, S91_R92delinsEP, N96_T97delinsNS, Q101R, A103L, L105V,K107_L110delinsRPDQ, V113_S116delinsAVDS, H118Y, H120D,Y122_A127delinsGCEPCGN, V129T, S131N, 135F_136VdelinsAI, W138R,Q140_H143delinsFSRF, A145_D148delinsAVKD, V150A, I152A,T156_T157delinsAA, E159_S161delinsGDA, K163A, E167A, V169_E170delinsYD,T173L, K176_R178delinsQEK, K180_A181 delinsGL, N183_F186delinsDVML, P198A201delinsRPSQ, K203 N204delinsSS, R208W, D210S, R212T, V214Q, G218P,Q220_E221delinsSA, V225_S228delinsATP, N230H, L238_R239delinsVA,Q241_S246delinsMLLRGA, K250D, N252_V254delinsALP, D256N,K259_A260delinsAA, K262G, T264_E267delinsSDQL, L269_V271delinsQAI,F275Y, F279_K280delinsVM, and Q282_S305delinsK.

Next, we conceptualized a sequential approach to engineer D1-variantswith D1L3 activity that starts with the transfer of multiple adjacentbuilding blocks (clusters), continues with the transfer of individualbuilding blocks, and ends with a transfer of individual amino acids orthe combination of multiple building blocks into new chimeric enzymes(FIG. 32). This approach reduces the number of D1-D1L3-chimera in theinitial screening.

To test our method, we designed a total of 19 D1-variants comprisingeither individual building blocks or clusters of building block clusterfrom D1L3 (FIG. 31). These D1-variants feature the following amino acidmutations:

-   1M_S22delinsMSRELAPLLLLLLSIHSALA,-   L23_A27delinsMRICS/I30_T32delinsVRS/E35_T36delinsES,-   M38_I47delinsQEDKNAMDVI, Q49_S52delinsKVIK/Y54C/I56_Q60delinsIILVM,-   V62_R63delinsIK/S65_K72delinsSNNRICPI/L74_N76delinsMEK,-   Q79_T84delinsRNSRRGIT, H86N/V88_V89delinsVI/E91_P92delinsSR,-   N96_S97delinsNT/R101Q/L103A/V105L,-   R107_Q110delinsKEKL/A113_S116delinsVKRS/Y118H/D120H,-   G122_N128delinsYQDGDA/T130S/N132S, A136_I137delinsFV,-   R139W/F141_F144delinsQSPH/E146_E149delinsAVKD/A151V/V153I/A157_A158d-   elinsTT/G160_A162delinsETS/A164K, A168E/Y170_D171delinsVE/L174T,-   Q177_K179delinsKHR/G181_L182delinsKA/D184_L187delinsNFIF,-   R199_Q202delinsPKKA/S204_S205delinsKN/W209R/S211D/T213R/Q215V/P219G/S221_A222delinsQE,    A226_P228delinsVKKS,-   H230NN238_A239delinsLR/M241_A246delinsQEIVSS/D250K/A252_P254delinsNS    V,-   N256D/A259_A260delinsKA/G262K/S264_L267delinsTEEE/Q269_I271delinsLDV,    and Y275F/V279_M280delinsFK/K282delinsQSSRAFTNSKKSVTLRKKTKSKRS.

Next, we cloned the cDNA into an expression vector, which wastransfected into HEK293 cells. Analysis of the cell supernatants showeddsDNA degradation by all samples (FIG. 33). Furthermore, we observedthat the transfer of building blocks (BB) 11 (see SEQ ID NO: 10), BB12-14 (see SEQ ID NO: 11), BB 26 (see SEQ ID NO: 12), BB 41-48 (see SEQID NO: 13), and BB 49 (see SEQ ID NO: 14) from D1L3 to D1 resulted inenzymes with increased chromatin degrading activity. All these chimericenzymes exhibited the same or more activity to degrade dsDNA substratesthan wild-type D1. The building blocks 11 (RNSRRGI in SEQ ID NO: 2) and49 (VKKS in SEQ ID NO: 2) from D1L3 contain R80/R81 and K227,respectively, which are not present in D1. The D1L3-BB cluster 41-48(PKKAWKNIRLRTDPRFVWLIGDQE from SEQ ID NO:2) features 5 additionalarginine and lysine residues than its counterpart in D1(RPSQWSSIRLWTSPTFQWLIPDSA of SEQ ID NO:1). These additional cationicamino acids may be responsible for the hyperactivity. The D1-buildingblocks 12-14 and 26 contain the amino acid sequences H86 to R95 and A136to V138 in SEQ ID NO: 1, which includes amino acid residues that arerequired for binding of the D1-inhibitor actin. Thus, replacement ofthese amino acid sequences with the respective building blocks fromD1L3, which do not interact with actin, likely generates actin-resistantvariants of D1. We now combined BB 11, 14, 26, 41-19 in one novelD1-variant (SEQ ID NO: 15). We observed that the combination of thesegain-of-function BBs increased the chromatin degrading of the D1 variantto levels of wild-type D1L3 (FIG. 33). Thus, the BB technology providesa robust method to generate hyperactive D1 variants.

Engineering DNase 1-Like 3 Variants through Building Block Technology

The BB cluster 60 to 62 of D1L3 includes the amino acid from itsC-terminal tail (FIG. 33). The tail starts after L281 (FIG. 34) andcomprises the amino acid sequence QSSRAFTNSKKSVTLRKKTKSKRS (SEQ IDNO:40), which includes a bi-partite NLS. D1L3 requires the C-terminaltail to degrade DNA within lipid vesicles including transfectionreagents and apoptotic bodies (7, 10, 28). Furthermore, the C-terminaltail is required for degradation of lipid- and cell-free chromatin byD1L3 (7). Both findings are based on a D1L3-variant that lacks the aminoacids after Q282 and is thus inactive in degrading these substrates.Wilbert et al. attached the C-terminal extension of D1L3 to theC-terminus of D1 and thus transferred the capacity to degrade DNA withinlipid vesicles to D1 (10, 28). Here, we employed our BB technology totransfer the C-terminus from D1L3 to D1 and vice versa. We used theconserved S272/D273/H274 motif, which is located in front of theterminal beta-sheet in D1 (FIG. 28), as a N-anchor and exchanged theterminal 3 building blocks between D1 and D1L3 (FIG. 34). This approachgenerated a D1-variant that features the C-terminal extension of D1L3due to the mutationsY275FN279_M280delinsFK/K282delinsQSSRAFTNSKKSVTLRKKTKSKRS (SEQ ID NO:16) and a truncated D1L3-variant due to the mutationsF275Y/F279_K280delinsVM/Q282_S305delinsK (SEQ ID NO: 17). We expressedboth variants and the wild-type enzymes in HEK293 cells andcharacterized the DNase activity in the supernatant. Unexpectedly, weobserved that the D1-variant with the C-terminus of D1L3 did not degradechromatin, while the D1L3-variant lacking the C-terminus of thewild-type enzyme was still able to cleave chromatin (FIG. 34). In fact,we observed an increased chromatin degrading activity with this D1L3variant. To corroborate this finding, we tested whether the truncatedvariant of D1L3 in vivo and observed that its expression of protectedfrom NET-mediated mortality of neutrophilic Dnase1^(−/−)Dnase1l3⁻⁻ mice(FIG. 34). In conclusion, the deletion of the C-terminal does notinhibit, but rather increases the function of D1L3 to degrade lipid-freeand cell-free chromatin.

Serum proteases, in particular plasmin, degrade D1L3, but not D1 (9).The sensitivity of D1L3 towards protease activity would limit its use inclinical practice. Glycosylated proteins display an increased protectionagainst proteases (32). Using software analysis, we could not identify aknown consensus sequence for glycosylation in human and murine D1L3.Software analysis of human D1 revealed 2 glycosylation sites, asobserved both in silico and in vitro (9). We hypothesized thatglycosylated variants of D1L3 may increase the half-life in circulationdue to an improved resistance towards serum proteases. A glycosylationsite requires a minimal consensus sequence of N-X-T/S. D1 contain twoknown N-glycosylation sites, N40 (N18 without signal peptide) and N128[(N106 without signal peptide, (9)]. The amino acids of theD1-glycosylation sites N40-X-T42 and N128-X-T130 correspond to theD38-X-N40 and A127-X-V129 in D1L3. Thus, a glycosylated version of D1L3needs to feature the amino acids mutations D38N, N40T, A127N, and V129T(see SEQ ID NO: 18). Instead of using conventional point mutations toinsert these amino acid substitutions, we used BB technology to transferthe D1-glysylation sites N40-X-T42, which lies within BB4 (MSNATLVSY inSEQ ID NO: 1), and N128-X-T130, which lies within the BB cluster 23-25(GCEPCGNDTFN in SEQ ID NO: 1), from D1 to D1L3 (FIG. 35). Thus, wegenerated two new D1L3 variants, which feature the mutationsQ36_I45delinsMSNATLVSYI (SEQ ID NO: 19) and Y122A127delinsGCEPCGNN129T/S131N (SEQ ID NO: 20), respectively. We expressedthese D1L3 variants along with the WT enzyme in HEK293 cells. Next, weanalyzed the supernatant by Western Blot using a polyclonal antibodytargeting an epitope in the C-terminal tail of the WT enzyme to detectthe WT enzyme as well as the D1L3 variants. As expected the D1L3variants showed bands of a higher molecular weight presumably toglycosylation, when compared to the WT enzyme (FIG. 35). D1L3 variantwith glycosylation at N40 or N128 retained their chromatin degradingactivity (FIG. 35). In conclusion, the data suggest that BB technologycan be used to transfer the glycosylation sites from D1 to D1L3, whichmay generate D1L3 variants with increased protease-resistance andhalf-life in patients.

Therapy with DNase1-Like 3 and a Variant Thereof

Finally, we tested the therapeutic effects of D1L3 in vivo. Histologicalanalysis of vascular clots of NETs show dense aggregates of chromatin(FIG. 36A), indicating that histone-free cleavage sites for D1 are rarein vivo. Thus, we hypothesized that an acute therapy with wild-type D1L3is more effective in degrading intravascular NETs than wild-type D1. Totest our hypothesis, we expressed Csf3 in Dnase1^(−/−)Dnase1l3^(−/−)deficient mice to induce vascular clots of NETs. Importantly, precedingexitus, mice with vascular occlusions by NETs develop hematuria andhypothermia, which can be detected non-invasively. Therefore, the animalmodel allows identifying and hence selecting mice, which have formedNETs, to test drug candidates for an acute therapy against NETs (FIG.36B). Here, we tested purified wild-type human D1 (dornase alpha; SEQ IDNO: 1), wild-type human D1L3 (SEQ ID NO: 2), and a hyperactive D1L3variant [D1L3^(v); (SEQ ID NO: 17)] for an acute therapy against NETs.We injected Csf3 into Dnase1^(−/−)Dnase1l3^(−/−) deficient mice. After72 hours, the first individual animals started to develop hypothermiaand hematuria. We used a drop in body temperature of 4° C. or more andthe presence of red urine as inclusion criteria. Mice, which developedthese phenotypes, were randomized into the different treatment groups.Animals received a single dose, intravenous injection via the tail veinof 1 mg/kg of D1, D1L3, or vehicle. After 30 minutes, the animals wereeuthanized to collect biosamples (blood and organs). Quantification ofclots of NETs in the vasculature of lungs showed that NETs occludedblood vessels in mice receiving vehicle or D1 therapy (FIG. 36D).Importantly, mice injected with D1L3 and D1L3^(v)showed no or few clotof NETs (FIG. 36D). In patients, circulating DNA has the size of DNA inmono-nucleosomes [180 base pairs, (33)], indicating that it is adegradation product of high-molecular weight DNA in chromatin.Circulating DNA is commonly used as a surrogate marker of intravascularNET formation, but can also derive from damaged and necrotic tissue(34). We quantified DNA in serum using a fluorescent DNA probe. Weobserved that the injection of D1L3 and D1L3^(v), but not D1 or vehicle,caused a strong increase in levels of circulating DNA (FIG. 36E).Isolation and visualization of circulating DNA showed multimers 180 basepair fragments, indicating the presence of mono- and oligo-nucleosomesin serum from in mice receiving the D1L3 therapy (FIG. 36F). Micereceiving D1L3^(v)showed only mono- and di-nucleosomes. The data confirmthe hyperactivity of D1L3^(v)in vivo. DNA isolates from serum of micereceiving D1 therapy showed faint DNA smears of various sizes, whereasno DNA could be isolated from serum of mice injected with vehicle (FIG.36F). Thus, D1L3 may be used, not only for prophylaxis, but also foracute therapy of diseases that associated with NETs.

We further explored the therapeutic application of D1L3^(v)in a model ofischemia-reperfusion injury (FIG. 37A). In brief, one testicle of malewild-type rats was subjected to testicular torsion as previouslydescribed by us (35). The procedure causes an ischemic tissue injury.After randomization and de-torsion of the testicle, the rats receivedthree daily injections of vehicle dornase alpha (D1, 1mg/kg), or apurified DNase1L3 variant (SEQ ID NO: 17, D1L3^(v), 1 mg/kg). After 7days the ischemic testicle and untreated testicle were collected. Thetherapy with the D1L3^(v), but not saline or D1, significantly reducedthe testicular atrophy (FIG. 37B). Moreover, therapy with the D1L3^(v),but not saline or D1, substantially reduced the tissue injury andmajority of animals no signs of injury (FIG. 37C). Collectively, thedata identify D1L3 as a potent therapy against ischemia-reperfusioninjury.

Drug Development of DNase1 Variants, DNase 1-Like 3 and Variants

Nearly 70% of all protein therapeutics are produced using ChineseHamster Ovary (CHO) (36). Indeed, wild-type DNase1 (dornase alpha) isproduced in CHO cells. Despite significant advantages, cell linedevelopment and large-scale production in CHO still remains a hugechallenge due a considerable degree of variability and no reliablemethods for predicting or modeling cell growth characteristics (36).Importantly, CHO cells were not able to stably produce hyperactivevariants of D1, which prevented their clinical manufacturing (12). Themanufacturing properties of D1L3 are unknown. Thus, we developed stableCHO cell lines producing wild-type D1 (SEQ ID NO: 1), a hyperactive D1variant (SEQ ID NO: 7), wild-type D1L3 (SEQ ID NO: 2) and a hyperactiveD1L3 variant (SEQ ID NO: 17). We cultured the cell lines in bioreactorsand observed a stable protein expression with wild-type D1 and the D1variant, with the latter having strongly increased expression levels(FIG. 38), illustrating the feasibility of producing a hyperactive D1variant in CHO. Unexpectedly, we observed only minor protein levels ofwild-type D1L3 and the D1L3 variant (FIG. 38), pointing towards a majorchallenge for clinical manufacturing D1L3 and variants thereof.

Due to high cell densities in large-scale bioreactors, cells compete forresources and undergo cellular stress leading to increased cell deathcalled apoptosis, a cell death program used by multi-cellular organisms,including mammals (37). D1L3 targets DNA in apoptotic bodies (7). Wespeculated that the low levels of D1L3 are due to scavenging of theenzyme by these apoptotic vesicles. To overcome these limitations, wetested the production of D1, D1L3 and their variants using the microbialexpression system, Pichia pastoris, an established, FDA approved andGenerally Recognized As Safe (GRAS) organism with protein secretoryabilities (38, 39). A key limitation of this approach is that proteinsproduced by P. pastoris are often hyperglycosylated and may result inenhanced immunogenicity (40). The composition of D1L3 and its variantare non-glycosylated and optimal for expression in P. pastoris. Wemutated the glycosylation sites in D1 and its variant at N40S and N128Srespectively to generate deglycosylated proteins (SEQ ID NO: 21, 23).Protein expression in P. pastoris is generally achieved by couplingproteins of interest to secretory signal peptides of various origins. Weomitted this step to avoid the generation of immunogenic chimericproteins. We speculated that the native signal peptide of D1 and/or D1L3is recognized by P. pastoris. After codon-optimization was performed, weexpressed the cDNA in P. pastoris (SEQ ID NO: 22, 24-26). We achievedexpression of D1 and the D1 variant (not shown). Importantly, thisapproach furthermore enabled the expression of D1L3 and the D1L3variant, with the latter showing increased expression levels (FIG. 38).We purified the proteins and confirmed that the D1L3 has increasedactivity (FIG. 38). In conclusion, the data suggest that usingnon-mammalian expression systems, such as P. pastoris, enables clinicalmanufacturing of D1L3 and variants thereof.

Discussion

Two DNA-degrading enzymes have been widely used therapeutically inexperimental models and patients. Streptodornase, a secreted DNase fromStreptococcus s p., has been tested in disease models of skin homograft,bladder clots, wound debridement, meningitis. DNase1 (includingpancreatic DNase1) has been used therapeutically in experimental modelsof cancer, cystic fibrosis, lupus, thrombosis, stroke, sepsis, lunginjury, atherosclerosis, viral infection, sickle cell disease,myocardial infarction, ear infection, wound healing, liver injury,endocarditis, liver infection, pancreatitis, primary graft dysfunction,limb ischemia reperfusion, kidney injury, blood clotting, asthma,alum-induced inflammation, hepatorenal injury. Streptodornase has alsobeen used to treat patients with pleural exudations, hemotorax,intrabiliary blood clots, post pneumatic anemia, ulcers,otolaryngological conditions, oral infections, minor injuries,sinusitis, post-operative rhinoplasties, infertility, bladder catheter,wound cleaning, skin reaction test. Importantly, DNase1 has been testedtherapeutically in patients with pneumococcal meningitis, gout, legulcers, cystic fibrosis, Kartegener's syndrome, asthma, lobaratelectasis, chronic bronchitis, bronchiectasis, lupus, primary cilliarydyskinesia, bronchiolitis, empyema, pleural infections, cancer, dry eyesdisease, lower respiratory tract infections, chronic hematomas,Alzheimer's disease, and obstructive pulmonary disease. In this example,we identified D1L3, variants thereof, as well as D1 variants as novelcandidates for a DNase therapy (FIG. 39).

Methods Detection of Chromatin Degrading Activity

To measure chromatin degrading activity, we mixed samples with nucleiisolated from HEK293 (1,500,000-3,000,000 per reaction), 20 mM Tris-HClpH 7.4, 2 mM MgCl₂, 2 mM CaCl₂, 50 mM NaCl and protease inhibitors.After incubating the samples for 1 hour at 37° C., they were heated upto 65° C. for 10 minutes. We proceeded with DNA isolation using theQIAamp DNA Blood Mini Kit (Qiagen) according to the manufacturer'sprotocol. The eluent was then loaded onto an agarose gel and the DNAfragments were separated by gel electrophoresis. The DNA fluorescence ofthe gels was recorded with a fluorescence scanner.

Further methods are described in Example 1.

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Sequences of wild-type DNases SEQ ID NO: 1Human DNase1 (P24855), Amino acid sequence (Signal Peptide; Mature Protein):MRGMKLLGALLALAALLQGAVSLKIAAFNIQTFGETKMSNATLVSYIVQILSRYDIALVQEVRDSHLTAVGKLLDNLNQDAPDTYHYVVSEPLGRNSYKERYLFVYRPDQVSAVDSYYYDDGCEPCGNDTFNREPAIVRFFSRFTEVREFAIVPLHAAPGDAVAEIDALYDVYLDVQEKWGLEDVMLMGDFNAGCSYVRPSQWSSIRLWTSPTFQWLIPDSADTTATPTHCAYDRIVVAGMLLRGAVVPDSALPFNFQAAYGLSDQLAQAISDHYPVEVMLK SEQ ID NO: 2Human DNase1L3 (Q13609), Amino acid sequence (Signal Peptide; Mature Protein):MSRELAPLLLLLLSIHSALAMRICSFNVRSFGESKQEDKNAMDVIVKVIKRCDIILVMEIKDSNNRICPILMEKLNRNSRRGITYNYVISSRLGRNTYKEQYAFLYKEKLVSVKRSYHYHDYQDGDADVFSREPFVVWFQSPHTAVKDFVIIPLHTTPETSVKEIDELVEVYTDVKHRWKAENFIFMGDFNAGCSYVPKKAWKNIRLRTDPRFVWLIGDQEDTTVKKSTNCAYDRIVLRGQEIVSSVVPKSNSVFDFQKAYKLTEEEALDVSDHFPVEFKLQSSRAFTNSKKSVTLRKKTKSKRS SEQ ID NO: 3Murine DNase1 (P49183): Amino acid sequence (Signal Peptide; Mature Protein):MRYTGLMGTLLTLVNLLQLAGTLRIAAFNIRTFGETKMSNATLSVYFVKILSRYDIAVIQEVRDSHLVAVGKLLDELNRDKPDTYRYVVSEPLGRKSYKEQYLFVYRPDQVSILDSYQYDDGCEPCGNDTFSREPAIVKFFSPYTEVQEFAIVPLHAAPTEAVSEIDALYDVYLDVWQKWGLEDIMFMGDFNAGCSYVTSSQWSSIRLRTSPIFQWLIPDSADTTVTSTHCAYDRIVVAGALLQAAVVPNSAVPFDFQAEYGLSNQLAEAISDHYPVEVTLRKI SEQ ID NO: 4Rat DNase1 (P21704), Amino acid sequence (Signal Peptide; Mature Protein):MRYTGLMGILLTLVNLLQLAATLRIAAFNIRTFGDTKMSNATLSSYIVKILSRYDIAVVQEVRDTHLVAVGKLLDELNRDIPDNYRYIISEPLGRKSYKEQYLFVYRPSQVSVLDSYHYDDGCEPCGNDTFSREPAIVKFFSPYTEVREFAIVPLHSAPTEAVSEIDALYDVYLDVRQKWGLEDIMFMGDFNAGCSYVTSSQWSSIRLRTSPIFQWLIPDSADTTATSTHCAYDRIVVAGALLQAAVVPSSAVPFDFQAEYRLINQMAEAISDHYPVEVTLRKT SEQ ID NO: 34Murine DNase1L3 (O55070): Amino acid sequence (Signal Peptide; Mature Protein):MSLHPASPRLASLLLFILALHDTLALRLCSFNVRSFGASKKENHEAMDIIVKIIKRCDLILLMEIKDSSNNICPMLMEKLNGNSRRSTTYNYVISSRLGRNTYKEQYAFVYKEKLVSVKIKYHYHDYQDGDTDVFSREPFVVWFHSPFTAVKDFVIVPLHTTPETSVKEIDELVDVYTDVRSQWKTENFIFMGDFNAGCSYVPKKAWQNIRLRTDPKFVWLIGDQEDTTVKKSTSCAYDRIVLCGQEIVNSVVPRSSGVFDFQKAYDLSEEEALDVSDHFPVEFKLQSSRAFTNNRKSVSLKKRKKGNRS SEQ ID NO: 35Rat DNase1L3 (O89107): Amino acid sequence (Signal Peptide; Mature Protein):MSLYPASPYLASLLLFILALHGALSLRLCSFNVRSFGESKKENHNAMDIIVKIIKRCDLILLMEIKDSNNNICPMLMEKLNGNSRRSTTYNYVISSRLGRNTYKEQYAFLYKEKLVSVKAKYLYHDYQDGDTDVFSREPFVVWFQAPFTAAKDFVIVPLHTTPETSVKEIDELADVYTDVRRRWKAENFIFMGDFNAGCSYVPKKAWKNIRLRTDPNFVWLIGDQEDTTVKKSTSCAYDRIVLRGQEIVNSVVPRSSGVFDFQKAYELSEEEALDVSDHFPVEFKLQSSRAFTNSRKSVSLKKKKKGSRS SEQ ID NO: 36Chimpanzee DNase1 (H2QAH1): Amino acid sequence (Signal Peptide; MatureProtein): MRSMKLLGALLALAALLQGAVSLKIAAFNIQTFGETKMSNATLVSYIVQILSRYDIALVQEVRDSHLTAVGKLLDNLNQDAPDTYHYVVSEPLGRNSYKERYLFVYRPDQVSAVDSYYYDDGCEPCGNDTFNREPAIVRFFSRFTEVREFAIVPLHAAPGDAVAEIDALYDVYLDVQEKWGLEDVMLMGDFNAGCSYVRPSQWSSIRLRTSPAFQWLIPDSADTTATPTHCAYDRIVVAGMLLQGAVVPDSALPFNFQAAYGLSDQLAQAISDHYPVEVMLK SEQ ID NO: 37Chimpanzee DNase1L3 (H2QMU7): Amino acid sequence (Signal Peptide; MatureProtein): MSRELTPLLLLLLSIHSTLALRICSFNVRSFGESKQEDQNAMDVIVKVIKRCDIILVMEIKDSNNRICPILMEKLNRNSRRGITYNYVISSRLGRNTYKEQYAFLYKEKLVSVKRSYHYHDYQDGDADVFSREPFVVWFQSPHTAVKDFVIIPLHTTPETSVKEIDELVEVYTDVKHRWKAENFIFMGDFNAGCSYVPKKAWKNIRLRTDPRFVWLIGDQEDTTVKKSTNCAYDRIVLRGQEIVSSVVPKSNSVFDFQKAYKLTEEEALDVSDHFPVEFKLQSSRAFTNSKKSVTLRKKTKSKRS SEQ ID NO: 38Human DNase1-like 1 (NM_006730): Amino acid sequence (Signal Peptide; MatureProtein): MHYPTALLFLILANGAQAFRICAFNAQRLTLAKVAREQVMDTLVRILARCDIMVLQEVVDSSGSAIPLLLRELNRFDGSGPYSTLSSPQLGRSTYMETYVYFYRSHKTQVLSSYVYNDEDDVFAREPFVAQFSLPSNVLPSLVLVPLHTTPKAVEKELNALYDVFLEVSQHWQSKDVILLGDFNADCASLTKKRLDKLELRTEPGFHWVIADGEDTTVRASTHCTYDRVVLHGERCRSLLHTAAAFDFPTSFQLTEEEALNISDHYPVEVELKLSQAHSVQPLSLTVLLL LSLLSPQLCPAASEQ ID NO: 39Human DNase1-like 2 (NM_001374): Amino acid sequence (Signal Peptide; MatureProtein): MGGPRALLAALWALEAAGTAALRIGAFNIQSFGDSKVSDPACGSIIAKILAGYDLALVQEVRDPDLSAVSALMEQINSVSEHEYSFVSSQPLGRDQYKEMYLFVYRKDAVSVVDTYLYPDPEDVFSREPFVVKFSAPGTGERAPPLPSRRALTPPPLPAAAQNLVLIPLHAAPHQAVAEIDALYDVYLDVIDKWGTDDMLFLGDFNADCSYVRAQDWAAIRLRSSEVFKWLIPDSADTTVGNSDCAYDRIVACGARLRRSLKPQSATVHDFQEEFGLDQTQALAISDHFP VEVTLKFHRC-terminal tail of human DNase1L3 SEQ ID NO: 33 Amino acid sequence:SSRAFTNSKKSVTLRKKTKSKRS Sequences of DNase1 variants SEQ ID NO: 5Amino acid sequence (Signal Peptide; Mature Protein):MRGMKLLGALLALAALLQGAVSLKIAAFNIRTFGETKMSNATLVSYIVKILSRYDIALVQEVRDSHLTAVGKLLDNLNRDAPDTYHYVVSEPLGRNSYKERYLFVYRPDQVSAVDSYYYDDGCEPCGNDTFNREPAIVRFFSRFTEVREFAIVPLHAAPGDAVAEIDALYDVYLDVQEKWGLEDVMLMGDFNAGCSYVRPSQWSSIRLRTSPTFQWLIPDSADTTATPTHCAYDRIVVAGMLLRGAVVPDSALPFNFQAAYGLSDQLAQAISDHYPVEVMLK SEQ ID NO: 6Amino acid sequence (Signal Peptide; Mature Protein):MRGMKLLGALLALAALLQGAVSLKIAAFNIRTFGETKMSNATLVSYIVKILSRYDIALVQEVRDSHLTAVGKLLDNLNRDAPDTYHYVVSEPLGRNSYKERYLFVYRPDQVSAVDSYYYDDGCEPCGNDTFNREPAIVRFFSRFTEVREFAIVPLHAAPGDAVAEIDALYDVYLDVKEKWGLEDVMLMGDFNAGCSYVRPSQWSSIRLRTSPTFQWLIPDSADTTATPTHCAYDRIVVAGMLLRGAVVPDSALPFNFQAAYKLSDQLAQAISDHYPVEVMLK SEQ ID NO: 7Amino acid sequence (Signal Peptide; Mature Protein):MRGMKLLGALLALAALLQGAVSLKIAAFNIRTFGETKMSNATLVSYIVQILSRYDIALVQEVRDSHLTAVGKLLDNLNQDAPDTYHYVVSEPLGRNSYKERYLFVYRPDQVSAVDSYYYDDGCEPCGNDTFNREPFIVRFFSRFTEVREFAIVPLHAAPGDAVAEIDALYDVYLDVQEKWGLEDVMLMGDFNAGCSYVRPSQWSSIRLWTSPTFQWLIPDSADTTAKPTHCAYDRIVVAGMLLRGAVVPDSALPFNFQAAYGLSDQLAQAISDHYPVEVMLK SEQ ID NO: 8Amino acid sequence (Signal Peptide; Mature Protein):MRGMKLLGALLALAALLQGAVSLKIAAFNIRTFGETKMSNATLVSYIVQILSRYDIALVQEVRDSHLTAVGKLLDNLNQDAPDTYHYVVSEPLGRNSYKERYLFVYRPDQVSAVDSYYYDDGCEPCGNDTFNREPAIVRFFSRFTEVREFAIVPLHAAPGDAVAEIDALYDVYLDVQEKWGLEDVMLMGDFNAGCSYVRPSQWSSIRLWTSPTFQWLIPDSADTTAKPTHCAYDRIVVAGMLLRGAVVPDSALPFNFQAAYGLSDQLAQAISDHYPVEVMLK SEQ ID NO: 9Amino acid sequence (Signal Peptide; Mature Protein):MRGMKLLGALLALAALLQGAVSLKIAAFNIQTFGETKMSNATLVSYIVQILSRYDIALVQEVRDSHLTAVGKLLDNLNQDAPDTYHYVVSEPLGRNSYKERYLFVYRPDQVSAVDSYYYDDGCEPCGNDTFNREPFIVRFFSRFTEVREFAIVPLHAAPGDAVKEIDALYDVYLDVQEKWGLEDVMLMGDFNAGCSYVRPSQWSSIRLWTSPTFQWLIPDSADTTATPTHCAYDRIVVAGMLLRGAVVPDSALPFNFQAAYGLSDQLAQAISDHYPVEVMLK SEQ ID NO: 10Amino acid sequence (Signal Peptide; Mature Protein):MRGMKLLGALLALAALLQGAVSLKIAAFNIQTFGETKMSNATLVSYIVQILSRYDIALVQEVRDSHLTAVGKLLDNLNRNSRRGITYHYVVSEPLGRNSYKERYLFVYRPDQVSAVDSYYYDDGCEPCGNDTFNREPAIVRFFSRFTEVREFAIVPLHAAPGDAVAEIDALYDVYLDVQEKWGLEDVMLMGDFNAGCSYVRPSQWSSIRLWTSPTFQWLIPDSADTTATPTHCAYDRIVVAGMLLRGAVVPDSALPFNFQAAYGLSDQLAQAISDHYPVEVMLK SEQ ID NO: 11Amino acid sequence (Signal Peptide; Mature Protein):MRGMKLLGALLALAALLQGAVSLKIAAFNIQTFGETKMSNATLVSYIVQILSRYDIALVQEVRDSHLTAVGKLLDNLNQDAPDTYNYVISSRLGRNSYKERYLFVYRPDQVSAVDSYYYDDGCEPCGNDTFNREPAIVRFFSRFTEVREFAIVPLHAAPGDAVAEIDALYDVYLDVQEKWGLEDVMLMGDFNAGCSYVRPSQWSSIRLWTSPTFQWLIPDSADTTATPTHCAYDRIVVAGMLLRGAVVPDSALPFNFQAAYGLSDQLAQAISDHYPVEVMLK SEQ ID NO: 12Amino acid sequence (Signal Peptide; Mature Protein):MRGMKLLGALLALAALLQGAVSLKIAAFNIQTFGETKMSNATLVSYIVQILSRYDIALVQEVRDSHLTAVGKLLDNLNQDAPDTYHYVVSEPLGRNSYKERYLFVYRPDQVSAVDSYYYDDGCEPCGNDTFNREPFVVRFFSRFTEVREFAIVPLHAAPGDAVAEIDALYDVYLDVQEKWGLEDVMLMGDFNAGCSYVRPSQWSSIRLWTSPTFQWLIPDSADTTATPTHCAYDRIVVAGMLLRGAVVPDSALPFNFQAAYGLSDQLAQAISDHYPVEVMLK SEQ ID NO: 13Amino acid sequence (Signal Peptide; Mature Protein):MRGMKLLGALLALAALLQGAVSLKIAAFNIQTFGETKMSNATLVSYIVQILSRYDIALVQEVRDSHLTAVGKLLDNLNQDAPDTYHYVVSEPLGRNSYKERYLFVYRPDQVSAVDSYYYDDGCEPCGNDTFNREPAIVRFFSRFTEVREFAIVPLHAAPGDAVAEIDALYDVYLDVQEKWGLEDVMLMGDFNAGCSYVPKKAWKNIRLRTDPRFVWLIGDQEDTTATPTHCAYDRIVVAGMLLRGAVVPDSALPFNFQAAYGLSDQLAQAISDHYPVEVMLK SEQ ID NO: 14Amino acid sequence (Signal Peptide; Mature Protein):MRGMKLLGALLALAALLQGAVSLKIAAFNIQTFGETKMSNATLVSYIVQILSRYDIALVQEVRDSHLTAVGKLLDNLNQDAPDTYHYVVSEPLGRNSYKERYLFVYRPDQVSAVDSYYYDDGCEPCGNDTFNREPAIVRFFSRFTEVREFAIVPLHAAPGDAVAEIDALYDVYLDVQEKWGLEDVMLMGDFNAGCSYVRPSQWSSIRLWTSPTFQWLIPDSADTTVKKSTHCAYDRIVVAGMLLRGAVVPDSALPFNFQAAYGLSDQLAQAISDHYPVEVMLK SEQ ID NO: 15Amino acid sequence (Signal Peptide; Mature Protein):MRGMKLLGALLALAALLQGAVSLKIAAFNIQTFGETKMSNATLVSYIVQILSRYDIALVQEVRDSHLTAVGKLLDNLNRNSRRGITYNYVISSRLGRNSYKERYLFVYRPDQVSAVDSYYYDDGCEPCGNDTFNREPFVVRFFSRFTEVREFAIVPLHAAPGDAVAEIDALYDVYLDVQEKWGLEDVMLMGDFNAGCSYVPKKAWKNIRLRTDPRFVWLIGDQEDTTVKKSTHCAYDRIVVAGMLLRGAVVPDSALPFNFQAAYGLSDQLAQAISDHYPVEVMLK SEQ ID NO: 16Amino acid sequence (Signal Peptide; Mature Protein):MRGMKLLGALLALAALLQGAVSLKIAAFNIQTFGETKMSNATLVSYIVQILSRYDIALVQEVRDSHLTAVGKLLDNLNQDAPDTYHYVVSEPLGRNSYKERYLFVYRPDQVSAVDSYYYDDGCEPCGNDTFNREPAIVRFFSRFTEVREFAIVPLHAAPGDAVAEIDALYDVYLDVQEKWGLEDVMLMGDFNAGCSYVRPSQWSSIRLWTSPTFQWLIPDSADTTATPTHCAYDRIVVAGMLLRGAVVPDSALPFNFQAAYGLSDQLAQAISDHFPVEFKLQSSRAFTNSKKSVTLRKKTKSKRS Sequences of DNase1L3 variants SEQ ID NO: 17Amino acid sequence (Signal Peptide; Mature Protein):MSRELAPLLLLLLSIHSALAMRICSFNVRSFGESKQEDKNAMDVIVKVIKRCDIILVMEIKDSNNRICPILMEKLNRNSRRGITYNYVISSRLGRNTYKEQYAFLYKEKLVSVKRSYHYHDYQDGDADVFSREPFVVWFQSPHTAVKDFVIIPLHTTPETSVKEIDELVEVYTDVKHRWKAENFIFMGDFNAGCSYVPKKAWKNIRLRTDPRFVWLIGDQEDTTVKKSTNCAYDRIVLRGQEIVSSVVPKSNSVFDFQKAYKLTEEEALDVSDHYPVEVMLK SEQ ID NO: 18Amino acid sequence (Signal Peptide; Mature Protein):MSRELAPLLLLLLSIHSALAMRICSFNVRSFGESKQENKTAMDVIVKVIKRCDIILVMEIKDSNNRICPILMEKLNRNSRRGITYNYVISSRLGRNTYKEQYAFLYKEKLVSVKRSYHYHDYQDGDNDTFSREPFVVWFQSPHTAVKDFVIIPLHTTPETSVKEIDELVEVYTDVKHRWKAENFIFMGDFNAGCSYVPKKAWKNIRLRTDPRFVWLIGDQEDTTVKKSTNCAYDRIVLRGQEIVSSVVPKSNSVFDFQKAYKLTEEEALDVSDHFPVEFKLQSSRAFTNSKKSVTLRKKTKSKRS SEQ ID NO: 19Amino acid sequence (Signal Peptide; Mature Protein):MSRELAPLLLLLLSIHSALAMRICSFNVRSFGESKMSNATLVSYIVKVIKRCDIILVMEIKDSNNRICPILMEKLNRNSRRGITYNYVISSRLGRNTYKEQYAFLYKEKLVSVKRSYHYHDYQDGDADVFSREPFVVWFQSPHTAVKDFVIIPLHTTPETSVKEIDELVEVYTDVKHRWKAENFIFMGDFNAGCSYVPKKAWKNIRLRTDPRFVWLIGDQEDTTVKKSTNCAYDRIVLRGQEIVSSVVPKSNSVFDFQKAYKLTEEEALDVSDHFPVEFKLQSSRAFTNSKKSVTLRKKTKSKRS SEQ ID NO: 20Amino acid sequence (Signal Peptide; Mature Protein):MSRELAPLLLLLLSIHSALAMRICSFNVRSFGESKQEDKNAMDVIVKVIKRCDIILVMEIKDSNNRICPILMEKLNRNSRRGITYNYVISSRLGRNTYKEQYAFLYKEKLVSVKRSYHYHDGCEPCGNDTFNREPFVVWFQSPHTAVKDFVIIPLHTTPETSVKEIDELVEVYTDVKHRWKAENFIFMGDFNAGCSYVPKKAWKNIRLRTDPRFVWLIGDQEDTTVKKSINCAYDRIVLRGQEIVSSVVPKSNSVFDFQKAYKLTEEEALDVSDHFPVEFKLQSSRAFTNSKKSVTLRKKTKSKRS Sequences used in Pichia pastoris SEQ ID NO: 21Amino acid sequence (Signal Peptide; Mature Protein):MRGMKLLGALLALAALLQGAVSLKIAAFNIQTFGETKMSSATLVSYIVQILSRYDIALVQEVRDSHLTAVGKLLDNLNQDAPDTYHYVVSEPLGRNSYKERYLFVYRPDQVSAVDSYYYDDGCEPCGSDTFNREPAIVRFFSRFTEVREFAIVPLHAAPGDAVAEIDALYDVYLDVQEKWGLEDVMLMGDFNAGCSYVRPSQWSSIRLWTSPTFQWLIPDSADTTATPTHCAYDRIVVAGMLLRGAVVPDSALPFNFQAAYGLSDQLAQAISDHYPVEVMLK SEQ ID NO: 22Codon-optimized cDNA of SEQ ID NO: 21 ( Start codon, Signal Peptide):ATGAGAGGTATGAAGCTCCTTGGTGCCCTCCTGGCTCTCGCTGCTCTCCTCCAAGGAGCAGTTTCTCTAAAGATTGCTGCTTTCAATATTCAAACCTTTGGCGAGACTAAAATGTCCTCGGCAACATTAGTATCTTACATTGTTCAGATCCTTTCTCGTTACGACATAGCTTTGGTCCAGGAGGTTAGAGACTCCCACTIGACTGCCGTCGGTAAGCTGCTAGACAATCTTAACCAAGACGCACCTGACACCTACCACTACGTTGTTAGTGAGCCTCTCGGTAGAAACTCCTACAAGGAGAGATACCTATTTGTCTACCGTCCAGATCAGGTGTCTGCTGTGGACTCTTACTACTATGACGACGGTTGTGAACCTTGTGGTTCAGACACCTTCAACAGAGAACCAGCTATCGTTAGATTTTTCTCCAGATTCACCGAGGTCAGAGAGTTCGCCATCGTTCCATTGCACGCTGCGCCTGGAGATGCAGTGGCCGAAATTGACGCTCTCTATGATGTCTACCTGGACGTTCAGGAAAAATGGGGTCTAGAAGATGTTATGCTGATGGGAGACTTCAACGCTGGTTGCTCTTACGTTAGGCCATCTCAATGGTCAAGCATCAGACTATGGACTTCCCCAACGTTCCAATGGCTTATTCCTGACTCCGCTGATACTACCGCCACTCCCACTCATTGTGCATATGACAGAATTGTTGTCGCTGGTATGCTACTGCGTGGAGCTGTCGTACCAGATTCTGCTCTGCCATTTAACTTCCAAGCCGCATATGGTCTTTCTGACCAACTGGCTCAAGCCATCTCTGATCACTACCCTGTGGAAGTTATGCTTAAG SEQ ID NO: 23Amino acid sequence (Signal Peptide; Mature Protein):MRGMKLLGALLALAALLQGAVSLKIAAFNIRTFGETKMSSATLVSYIVQILSRYDIALVQEVRDSHLTAVGKLLDNLNQDAPDTYHYVVSEPLGRNSYKERYLFVYRPDQVSAVDSYYYDDGCEPCGSDTFNREPFIVRFFSRFTEVREFAIVPLHAAPGDAVAEIDALYDVYLDVQEKWGLEDVMLMGDFNAGCSYVRPSQWSSIRLWTSPTFQWLIPDSADTTAKPTHCAYDRIVVAGMLLRGAVVPDSALPFNFQAAYGLSDQLAQAISDHYPVEVMLK SEQ ID NO: 24Codon-optimized cDNA of SEQ ID NO: 23 ( Start codon, Signal Peptide):ATGAGAGGTATGAAGCTCCTTGGTGCCCTCCTGGCTCTCGCTGCTCTCCTCCAAGGAGCAGTTTCTCTAAAGATTGCTGCTTTCAATATTAGAACCTTTGGCGAGACTAAAATGTCCTCGGCAACATTAGTATCTTACATTGTTCAGATCCTTTCTCGTTACGACATAGCTTTGGTCCAGGAGGTTAGAGACTCCCACTIGACTGCCGTCGGTAAGCTGCTAGACAATCTTAACCAAGACGCACCTGACACCTACCACTACGTTGTTAGTGAGCCTCTCGGTAGAAACTCCTACAAGGAGAGATACCTATTTGTCTACCGTCCAGATCAGGTGTCTGCTGTGGACTCTTACTACTATGACGACGGTTGTGAACCTTGTGGTTCAGACACCTTCAACAGAGAACCATTCATCGTTAGATTTTTCTCCAGATTCACCGAGGTCAGAGAGTTCGCCATCGTTCCATTGCACGCTGCGCCTGGAGATGCAGTGGCCGAAATTGACGCTCTCTATGATGTCTACCTGGACGTTCAGGAAAAATGGGGTCTAGAAGATGTTATGCTGATGGGAGACTTCAACGCTGGTTGCTCTTACGTTAGGCCATCTCAATGGTCAAGCATCAGACTATGGACTTCCCCAACGTTCCAATGGCTTATTCCTGACTCCGCTGATACTACCGCCAAGCCCACTCATTGTGCATATGACAGAATTGTTGTCGCTGGTATGCTACTGCGTGGAGCTGTCGTACCAGATTCTGCTCTGCCATTTAACTTCCAAGCCGCATATGGTCTTTCTGACCAACTGGCTCAAGCCATCTCTGATCACTACCCTGTGGAAGTTATGCTTAAG SEQ ID NO: 25Codon-optimized cDNA of SEQ ID NO: 2 ( Start codon, Signal Peptide):ATGTCTAGAGAGCTTGCTCCACTCCTCCTACTCCTTCTTAGTATCCACTCTGCACTCGCCATGAGAATTTGTAGCTTCAATGTAAGATCCTTCGGTGAATCTAAGCAGGAGGACAAAAACGCTATGGACGTTATTGTGAAGGTCATTAAGAGATGTGATATCATTCTAGTTATGGAGATCAAGGACTCTAACAACAGAATTTGCCCCATCCTTATGGAAAAGCTGAATAGAAACTCTAGAAGAGGAATTACTTACAACTACGTTATCTCTTCTAGGCTGGGTAGAAACACTTACAAGGAGCAATATGCATTTCTATACAAGGAAAAGCTAGTTTCCGTTAAGCGTTCTTACCACTATCATGACTACCAAGACGGCGATGCTGACGTTTTCTCCAGAGAACCATTCGTTGTTTGGTTTCAGTCTCCTCACACCGCTGTTAAGGACTTCGTGATTATCCCACTACACACTACGCCAGAAACCTCTGTCAAGGAAATAGATGAACTTGTTGAAGTTTACACCGACGTGAAGCACAGATGGAAGGCCGAGAATTTCATTTTCATGGGTGATTTCAACGCCGGATGCTCATATGTCCCTAAAAAGGCTTGGAAAAACATCCGTTTGAGAACCGATCCTAGATTTGTCTGGCTCATCGGTGACCAAGAGGACACCACAGTCAAAAAGTCTACCAACTGTGCTTACGACAGAATTGTTCTGCGTGGTCAGGAGATTGTTTCATCTGTTGTCCCAAAGTCCAACTCCGTCTTTGATTTCCAAAAGGCTTACAAACTGACTGAGGAGGAAGCTTTAGACGTGTCCGACCACTTCCCTGTAGAGTTTAAGCTGCAATCCTCCAGAGCATTCACTAACTCTAAAAAGTCAGTCACTTTGCGTAAAAAGACTAAGTCTAAGAGATCG SEQ ID NO: 26Codon-optimized cDNA of SEQ ID NO: 17 ( Start codon, Signal Peptide):ATGTCTAGAGAGCTTGCTCCACTCCTCCTACTCCTTCTTAGTATCCACTCTGCACTCGCCATGAGAATTTGTAGCTTCAATGTAAGATCCTTCGGTGAATCTAAGCAGGAGGACAAAAACGCTATGGACGTTATTGTGAAGGTCATTAAGAGATGTGATATCATTCTAGTTATGGAGATCAAGGACTCTAACAACAGAATTTGCCCCATCCTTATGGAAAAGCTGAATAGAAACTCTAGAAGAGGAATTACTTACAACTACGTTATCTCTTCTAGGCTGGGTAGAAACACTTACAAGGAGCAATATGCATTTCTATACAAGGAAAAGCTAGTTTCCGTTAAGCGTTCTTACCACTATCATGACTACCAAGACGGCGATGCTGACGTTTTCTCCAGAGAACCATTCGTTGTTTGGTTTCAGTCTCCTCACACCGCTGTTAAGGACTTCGTGATTATCCCACTACACACTACGCCAGAAACCTCTGTCAAGGAAATAGATGAACTTGTTGAAGTTTACACCGACGTGAAGCACAGATGGAAGGCCGAGAATTTCATTTTCATGGGTGATTTCAACGCCGGATGCTCATATGTCCCTAAAAAGGCTTGGAAAAACATCCGTTTGAGAACCGATCCTAGATTTGTCTGGCTCATCGGTGACCAAGAGGACACCACAGTCAAAAAGTCTACCAACTGTGCTTACGACAGAATTGTTCTGCGTGGTCAGGAGATTGTTTCATCTGTTGTCCCAAAGTCCAACTCCGTCTTTGATTTCCAAAAGGCTTACAAACTGACTGAGGAGGAAGCTTTAGACGTGTCCGACCACTACCCTGTAGAGGTCATGTTGAAG

1-157. (canceled)
 158. A method for treating a human subject having anintravascular accumulation of neutrophil extracellular traps (NETs), themethod comprising systemically administering an effective amount of aDNase1-like 3 (D1L3) enzyme comprising an amino acid sequence having atleast 90% sequence identity to amino acids 21 to 305 of SEQ ID NO: 2 tosaid subject, wherein the subject has a condition selected from: asevere inflammatory disease selected from acute respiratory distresssyndrome (ARDS), sepsis, and a viral infection; partially or fullyoccluded blood vessels; thrombotic microangiopathy (TMA); disseminatedintravascular coagulation (DIC); venous thromboembolism; leukemia; andtumor metastasis.
 159. The method of claim 158, wherein the D1L3 enzymecomprises a fusion to a carrier protein.
 160. The method of claim 159,wherein the carrier protein is albumin.
 161. (canceled)
 162. The methodof claim 158, wherein the D1L3 enzyme comprises an amino acid sequencehaving at least 95% sequence identity to amino acids 21 to 305 of theamino acid_sequence of SEQ ID NO:
 2. 163. The method of claim 158,wherein the D1L3 enzyme comprises a deletion of NLS2.
 164. The method ofclaim 158, wherein the D1L3 enzyme comprises a deletion of all or partof the C-terminal tail defined by SEQ ID NO:
 33. 165. The method ofclaim 158, wherein the D1L3 enzyme_is administered by intradermal,intramuscular, subcutaneous, intraperitoneal, intravenous, orintraarterial administration.
 166. (canceled)
 167. The method of claim158, wherein the subject has neutrophilia.
 168. The method of claim 158,wherein the subject has a severe inflammatory disease.
 169. The methodof claim 168, wherein the subject has sepsis.
 170. The method of claim168, wherein the subject has acute respiratory distress syndrome (ARDS).171. The method of claim 158, wherein the subject has partially or fullyoccluded blood vessels.
 172. The method of claim 171, wherein thepartially or fully occluded blood vessels are in the lung.
 173. Themethod of claim 171, wherein the occlusions comprise entrappederythrocytes and leukocytes. 174.-175 (canceled)
 176. The method ofclaim 158, wherein the subject has thrombotic microangiopathy (TMA).177. The method of claim 158, wherein the subject has disseminatedintravascular coagulation (DIC).
 178. The method of claim 158, whereinthe subject has venous thromboembolism.
 179. The method of claim 158,wherein the administration results in higher levels of circulating mono-and di-nucleosomes.
 180. (canceled)
 181. The method of claim 158,wherein the subject has leukemia.
 182. The method of claim 158, whereinthe subject has tumor metastasis.
 183. The method of claim 168, whereinthe subject has a viral infection.